Nano-sized particles comprising multi-headed amphiphiles for targeted drug delivery

ABSTRACT

Nano-sized particles are provided comprising at least one multi-headed amphiphilic compound, in which at least one headgroup of said multi-headed amphiphilic compound is selectively cleavable or contains a selectively cleavable group, and at least one biologically active agent, which is both encapsulated within the nano-particle and non-covalently associated thereto.

FIELD OF THE INVENTION

The present invention is in the field of drug delivery and, moreparticularly, relates to nano-sized particles comprising at least onemulti-headed amphiphilic compound and a biologically active agent.

BACKGROUND ART

The use of proteins, peptides and polynucleotides such as DNA and RNA(including small interference (si) RNA) in therapy or in preventivemedicine is limited because they are generally impermeable throughvarious biological barriers (e.g., blood-brain barrier (BBB) andmembrane barriers of the circulatory system, intestinal track, skin andlungs) and sensitive to proteolytic enzymes, thus not surviving thepassage from the site of administration to the site of action. Theselimitations result in poor pharmacokinetics (PK), preventing or limitingtheir use in the treatment of neurological diseases and in diseases inother organs of the body.

Many drugs and biologically active molecules cannot penetrate the BBBand thus require direct administration into the CNS tissue or thecerebral spinal fluid (CSF) in order to achieve a biological ortherapeutic effect. Even direct administration into a particular CNSsite is often limited due to poor diffusion of the active agent becauseof local absorption/adsorption into the CNS matrix. Present modalitiesfor drug delivery through the BBB entail disruption of the BBB by, forexample, osmotic means (hyperosmotic solutions) or biochemical means(e.g., use of vasoactive substances such as. bradykinin), processes withserious side effects.

In order to fulfill the therapeutic potential of peptides, proteins andnucleotides and other agents with poor PK, a non invasive deliverymethod is required that will distribute the agent at the desired area ofthe target site (e.g., a wide area of an organ such as the brain), willhave good blood circulatory lifetime for the delivery platform, willpenetrate through biological barriers and will have a selectivedisruption mechanism.

Small interference RNAs (siRNAs) are an example for polynucleotideswhich would have a highly promising therapeutic potential if only theirPK could be improved. RNA interference is a powerful strategy to inhibitgene expression through specific mRNA degradation mediated by siRNAs.However, in vivo application of siRNAs is severely limited by theirinstability and poor delivery to target cells and target tissues. siRNAscould be an alternative therapy of glioblastoma, a brain tumor highlyresistant to chemotherapy and radiotherapy. Gene silencing is apromising approach for inhibiting the proliferation of this type oftumor and several target genes may be considered for this therapeuticstrategy, such as epidermal growth factor receptor variant III, which isexpressed in 40-50% of gliomas, and the phosphoinositide 3-kinase(PI3K)/Akt pathway, which plays a crucial role in medulloblastomabiology. Targeting of such oncogenic pathways can be achieved by genesilencing with RNA interference. However, before RNA interference can beexploited for brain tumor therapy, several obstacles have to beovercome, such as the instability of siRNAs in the blood stream andtheir impermeability through the BBB.

An efficient delivery system for proteins, peptides, polynucleotides andother biologically active agents should protect the agents while theyare being transported, allow them to pass intact through biologicalbarriers such as the BBB, and target them to the site of action by amechanism that releases them specifically at that site. In order toachieve such performance, such a delivery system should preferablycomprise nano-sized drug carriers which are stable in biological fluids,penetrate intact various biological membranes and have a selectivedisruption mechanism. In addition, such a carrier should be able toencapsulate significant amounts of the active agent whereby manymolecules per vesicle or carrier are targeted to a particular site ororgan. There are, however, no currently efficient delivery systemswherein all these necessary properties are combined within one deliverysystem.

Complexation of the anionic carboxyfluorescein (CF) with single headedamphiphiles of opposite charge in cationic vesicles, formed by mixingsingle-tailed cationic and anionic surfactants has been reported (Danoffet al. 2007). Wang et al. (2006) disclose complexation of the anionic CFwith bilayered vesicles formed from cetyl trimethylammonium tosylate(CTAT) and sodium dodecylbenzenesulfonate (SDBS). The CTAT-rich(cationic) vesicles were shown to capture the CF with high efficiency(22%). The ability of these vesicles to capture and hold dyes is veryhigh (>20%) when the excess charge of the vesicle bilayer is opposite tothat of the solute (i.e., CTAT-rich vesicles capture anionic solutesvery efficiently, whereas SDBS-rich vesicles efficiently capturecationic solutes).

U.S. Pat. No. 6,358,523 discloses macromolecule-lipid complexes,macromolecule targeting and delivery to various biological systems.

WO 02/055011 and WO 03/047499, both of the same applicant, discloseamphiphilic derivatives composed of at least one fatty acid chainderived from natural vegetable oils such as vernonia oil, lesquerellaoil and castor oil, in which functional groups such as epoxy, hydroxyand double bonds were modified into polar and ionic headgroups. Theamphiphiles of WO 02/055011 and WO 03/047499 comprise one or more ionicor polar headgroups and at least one hydrogen-bonding group locatedeither within said headgroup and/or in close proximity thereto. Theseamphiphiles are capable of spontaneously forming vesicles and micellesowing to their polar and ionic headgroups.

WO 03/047499 discloses bolaamphiphiles (vesicle-forming amphiphiliccompounds bearing two headgroups), having at least one headgroupcontaining a selectively cleavable group or moiety such as a residue ofa choline or phenylalanine derivative. The cleavable group or moiety iscleaved and the vesicles disrupt and release their load under selectiveconditions, which include change of chemical, physical or biologicalenvironment. These vesicles are preferably cleaved enzymatically in abiological environment such as the brain or the blood. The vesicles orliposomes made from these amphiphilic compounds are highly stable,beyond what is achievable with the lipids and surfactants used in thecurrent state of the art, and suitable for delivery of a therapeuticsubstance or a diagnostic agent specifically to a target organ ortissue.

The prior art does not emphasize the benefits of using multi-headedamphiphiles for targeted delivery. Simultaneous complexation andencapsulation of small molecules and macromolecules such as peptides,proteins and nucleotides within vesicles of bolaamphiphiles ormulti-headed amphiphiles bearing selectively cleavable groups, is notdisclosed in the prior art either. However, it is the use of suchmulti-headed amphiphiles and particularly bolaamphiphiles that canachieve the desired combination.

SUMMARY OF INVENTION

In one aspect, the present invention relates to a nano-sized particlecomprising at least one multi-headed amphiphilic compound, in which atleast one headgroup of said multi-headed amphiphilic compound isselectively cleavable or contains a selectively cleavable group, and atleast one biologically active agent, which is both encapsulated withinthe nanoparticle and non-covalently associated thereto.

The nanoparticles of the invention are useful for delivery of thebiologically active agent to a target organ or tissue.

Thus, in another aspect, the present invention relates to apharmaceutical composition comprising nano-sized particles of theinvention and a pharmaceutically acceptable carrier.

Depending on the biologically active agent comprised within thenanoparticles of the invention, the nanoparticles or the pharmaceuticalcomposition comprising them can be used for treatment or diagnosis ofdiseases or disorders selected from: (i) diseases or disordersassociated with the central nervous system (CNS), in particularneurological and/or neurodegenerative diseases or disorders such asParkinson's disease, Alzheimer's disease or multiple sclerosis; (ii)cancer such as breast cancer and brain tumors; (iii) diabetes; (iv)immunodeficiency diseases; and (v) viral and bacterial infections.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the analgesic effect as percent of the maximalpossible effect (MPE) in a hot plate test conducted on mice treated withmorphine (5 mg/kg) (gray column), Derivative 4-nanoparticles loaded with5 mg/kg leu-enkephaime (Hatched column), empty Derivative 4-nanoparticle20 mg/kg (empty column), and free leu-enkephalin (20 mg/kg) (dottedcolumn). The values are means±SEM of 5 mice.

FIG. 2 is a graph showing the analgesic effect as percent of the maximalpossible effect (MPE) in a hot plate test conducted on mice treated withfree leu-enkephalin (25 mg/kg) (dotted column), Derivative4-nanoparticles loaded with 25 mg/kg leu-enkephalin (Hatched column),and DSPC liposomes loaded with leu-enkephalin (50 mg/kg) (waves). Thevalues are means±SEM of 5 mice.

FIG. 3 is a graph showing the brain uptake of carboxyfluoresceindelivered in nanoparticles comprising Derivative 4 and PEG₂₀₀₀-vernoniaderivatives. Dotted column: free CF (0.333 mg/Kg), column with waves:formulation containing Derivative 4/cholesterol/cholesterylhemisuccinate/PEG(202) nanoparticles (10 mg/kg) loaded with CF (0.333mg/Kg).

FIGS. 4A-4D are graphs showing the distribution of i.v. administeredcarboxyfluorescein in the brain (4A), heart (4B), lungs (4C) and kidneys(4D). Hatched: mice injected with a formulation containing the basicnanoparticles (10 mg/kg) loaded with 0.2 mg/ml CF; grid: mice were i.v.injected with a formulation containing chitosan-nanoparticles (10 mg/kg)loaded with 0.2 mg/ml CF pre-injected with pyridostigmine (0.5 mg/kg);and horizontal lines: mice i.v. injected with a formulation containingchitosan-nanoparticles loaded with CF without pre-injected ofpyridostigmine.

FIGS. 5A-5D are graphs showing the distribution of orally administeredcarboxyfluorescein in the brain (5A), heart (5B), lungs (5C) and kidneys(5D). Hatched: mice gavaged with a formulation containing the basicnanoparticles (10 mg/kg) loaded with 0.2 mg/ml CF; grid: mice weregavaged with a formulation containing chitosan-nanoparticles (10 mg/kg)loaded with 0.2 mg/ml CF pre-injected with pyridostigmine (0.5 mg/kg);and horizontal lines: mice gavaged with a formulation containingchitosan-nanoparticles loaded with CF without administration ofpyridostigmine.

FIG. 6 is a graph showing the analgesic effect as percent of the maximalpossible effect (MPE) in a hot plate test conducted on mice treated withvarious concentrations of leu-enkephalin. Gray: mice treated withmorphine (5 mg/kg); hatched: mice treated with Derivative4-nanoparticles loaded with 20 mg/kg leu-enkephaime; grids: mice treatedwith Derivative 4-nanoparticles loaded with 10 mg/kg leu-enkephaime;lines: mice treated with Derivative 4-nanoparticles loaded with 5 mg/kgleu-enkephaime; and dotted: mice treated with free leu-enkephalin (20mg/kg). The values are means±SEM of 5 mice.

FIG. 7 is a graph showing the analgesic effect as percent of the maximalpossible effect (MPE) in a hot plate test conducted on mice treatedleu-enkephalin delivered in various nanoparticles. Gray: mice treatedwith morphine (5 mg/kg); lines: mice i.v. injected with Derivative1+Derivative 4 nanoparticles (20 mg/kg) loaded with 5 mg/kgleu-enkephalin; group; horizontal lines: mice pre-injected withpyridostigmine and then i.v. injected with Derivative 1+Derivative 4nanoparticles (20 mg/kg) loaded with 5 mg/kg leu-enkephalin; group;dotted: mice treated with free leu-enkephalin (20 mg/kg). The values aremeans±SEM of 5 mice.

DETAILED DESCRIPTION OF THE INVENTION

It has been found by the present inventors that when multi-headedamphiphiles, particularly double-headed amphiphiles also termed“bolaamphiphiles”, comprising cationic headgroups were mixed with markermolecules with opposite anionic carboxylic charge (e.g.,carboxyfluorescein (CF)), vesicles were formed with a very highencapsulation efficiency than would be expected from vesicle size: 10 to30% instead of 5%. Good encapsulation efficiency for a given activeagent was obtained when bolaamphiphiles such as those disclosed in WO03/047499 of the same inventors, incorporated herein by reference as iffully described herein, were used for the preparation of nano-sizedvesicles in the presence of active agent solutes selected from peptides,proteins, polynucleotides or non-polymeric molecules. Surprisinglyefficient encapsulation occurred when the net ionic charges on theamphiphiles' head group and on the encapsulated molecule were opposite(e.g., cationic versus anionic). The unexpected high loading efficiencycould not be attributed solely to encapsulation of the peptides,proteins, polynucleotides or non-polymeric molecules within thevesicles' core, and other kind of interactions that associated themolecules to the vesicles had to be considered.

In other experiments performed by the inventors, when molecules withanionic carboxylic groups such as cholesterol hemisuccinate were mixedwith cationic bolaamphiphiles, stable vesicles were formed whereincholesterol hemisuccinate was shown to be taken up in the outer surfaceof the vesicles. At the same time, the use of cholesterol hemisuccinatereduced the percentage encapsulation of CF, indicating that CF may forma counter ion to the bolaamphiphile headgroups on the inside and outsidesurfaces of the vesicular membrane. The fact that the encapsulationefficiency of CF was still relatively high in the presence ofcholesterol hemisuccinate suggests that CF may be associated with thevesicles in some other manners besides being encapsulated within thevesicle core.

It is assumed that non-covalent interactions such as ionic interactionsbetween the solute and the oppositely charged headgroups result inattachment of solutes onto the vesicles' inner and outer surface. Inaddition, it is believed that some solute molecules may be embedded orimmersed in the vesicular membrane as well as within the core. Thus, thedelivery platform obtained is probably a nano-sized particle comprisingamphiphiles and active agent molecules, which are associated with theamphiphiles by way of encapsulation within vesicles formed from theamphiphiles, ionic interactions of oppositely charged groups and/orother non-covalent interactions, as well as immersion or incorporationwithin the vesicular matrix.

It was further found by the present inventors that for properly chosenheadgroups and amphiphiles, nano-sized particles were obtained whichpossessed excellent targeting delivery properties after i.v. and oraladministration. Proper headgroups were headgroups that could serve assubstrates for enzymes found in significant concentrations at the targetsite, and could enhance penetration through different biologicalbarriers.

The present inventors further found that use of amphiphiles withcationic headgroups such as choline ester headgroups, improvedpenetration of the vesicles made therefrom through biological bathers.Penetration of nanoparticles via the BBB was shown to be accelerated byproviding them with cationic surface groups.

It is well known, however, that cationic particles are cleared from theblood circulation within a period of less than one hour. The presentinventors have surprisingly found that particles comprising nano-sizedvesicles (as a non limiting example ˜80 nm) having a monolayer membranesignificantly slowed down the rapid particles clearance.

The present inventors also found that attaching certain additives suchas chitosan, chitosan derivatives and polyamines to the nanoparticlessurface enhanced penetration through the intestinal tract as well asother biological barriers. These additives proved particularly useful inoral dosage forms for delivery to the CNS or other organs such as theheart, muscles or lungs.

Thus, in one aspect, the present invention provides a nano-sizedparticle comprising at least one multi-headed amphiphilic compound, inwhich at least one headgroup of said multi-headed amphiphilic compoundis selectively cleavable or contains a selectively cleavable group, andat least one biologically active agent, which is both encapsulatedwithin the nano-sized particle and non-covalently associated thereto.

Non-covalent interactions, which may exists include, but are not limitedto, ionic and polar interactions, hydrogen bonding, electrostaticforces, hydrophobic interactions and Van der Waals forces.

In particular embodiments, the biologically active agent is associatedto the nano-sized particle via ionic interactions.

In other particular embodiments, the biologically active agent forms asalt complex with the nanoparticle. In a more particular embodiment theactive agent is associated with the nanoparticle via ionic interactionsbetween the multi-headed amphiphilic compound and oppositely chargedgroups of the active agent itself.

The nano-sized particle of the invention (also termed herein“nanoparticle”) may comprise molecules of non-encapsulated active agentsthat are embedded or immersed or incorporated in its matrix. Thus, incertain embodiments, the nano-sized particle comprises an amphiphiliccompound and a biologically active agent encapsulated therein,non-covalently associated thereto and, in addition, incorporated orembedded therein.

The nano-sized particles most often are in the form of vesicles orliposomes formed from the multi-headed amphiphiles having a core (whichmay be liquid or solid or gel) and a membrane surrounding the core, madeat least in part from these amphiphiles.

In particular embodiments, the nano-vesicles have an outer diameter ofless than 500 nm, preferably less than 150 nm. Such vesicles orliposomes may encapsulate within their core the active agent, which inparticular embodiments is selected from peptides, proteins, nucleotidesand or non-polymeric agents. The active agent in most cases is alsoassociated via one or more non-covalent interactions to the vesicularmembrane on the outer surface and/or the inner surface, optionally aspendant decorating the outer or inner surface, and may further beincorporated into the membrane surrounding the core. Particularly,biologically active peptides, proteins, nucleotides or non-polymericagents that have a net electric charge, may associate ionically withoppositely charged headgroups on the vesicle surface and/or form saltcomplexes therewith.

Nano-sized particles wherein the biologically active molecules arecomplexed with the vesicles are also referred to herein as “vesicularcomplexes”.

Complexation of multi-headed amphiphiles with the active agent may beobtained by way of forming salt complexes of the ionic headgroups andthe active agents. Formation of such salt complexes may influence thefinal size of the nanoparticles and their morphology. Such complexationincreases the effectiveness of the encapsulation process and impartssurface properties to the nanoparticles that would influence theirpharmacokinetics, bioavailability and targeted drug delivery properties.

The hydrophobic moieties or other groups of the active agent may alsointeract by secondary forces with the different components of the multiheaded amphiphiles, for example by hydrophobic-hydrophobic interactions.

The multi-headed amphiphilic compound, herein sometimes also termed“amphiphile” or “amphipathic compound”, constitutes the infrastructureof the nano-sized delivery system of the invention, and its structureand chemical properties determines to a great extent the stability andefficiency of delivery.

In a particular embodiment, the multi-headed amphiphiles is abolaamphiphile.

As used herein, the term “headgroup” is interpreted in the context of anamphiphilic compound and refers to a polar or ionic group attached tothe aliphatic chain of the amphiphile, either directly or indirectly(e.g., via a linker), and promotes or supports spontaneousself-aggregation of the amphiphiles in aqueous media.

When amphiphilic compounds are mixed with water, the polar or chargedregions and the non-polar regions (aliphatic chains) of the amphiphilesexperience conflicting tendencies; the polar or charged hydrophilicregions interact favorably with the solvent and tend to dissolve, butthe non-polar, hydrophobic regions have the opposite tendency, to avoidcontact with the water. The non-polar regions of the amphiphiles clustertogether to present the smallest hydrophobic area to the solvent, andthe polar regions are arranged to maximize their interactions with theaqueous solvent.

Ionic groups in amphiphiles often function as headgroups, however notall polar groups are headgroups. A polar group is a head group when itsupports (spontaneous) self-aggregation of the amphiphiles. This occurswhen the polar group has sufficient water solubility or attractivepowers. Thus, amides (—CO—NH—), epoxies, ethers (e.g., ethylene oxide)and even single hydroxyl groups are not sufficiently water attracting orsolubilizing to be considered headgroups. Sugars, however, with multiplehydroxyl groups and polyethylene glycols or polyethylene oxides withmultiple ethylene oxide groups, are highly water attracting with a largedegree of water of hydration, and function as headgroups. In certainembodiments of the invention, the nanoparticles comprise amphiphiliccompounds that have polar headgroups.

The amphiphiles used according to the invention have a low criticalaggregation concentration (CAC), preferably of less than 10⁻⁴, morepreferably less than 10⁻⁵, most preferably less than 10⁻⁶ moles.

For convenience, the term “selectively cleavable head group” shall beused throughout the description to denote both a headgroup that iscleaved under selective conditions and a headgroup containing removablegroup or moiety that is cleaved under selective conditions, wherein saidselective conditions include change of chemical, physical or biologicalenvironment such as, but not limited to, change of pH or temperature,oxidative or reducing conditions, and/or enzymatic conditions. The term“removable group” denotes a specific functional group within theselectively cleavable group or moiety that is removed from the moleculewhen the cleavage occurs, often together with the linker that connectsit to the hydrophobic chain.

In certain embodiments of the invention, the selectively cleavableheadgroup is cleaved enzymatically in a biological environment,particularly in the brain or blood, by degradatives enzymes such ashydrolases, esterases, phosphatases, oxidases, decarboxylases,deaminases and isomerases, some of which are restricted to the brain orexist also in the brain and in the periphery. Examples of such enzymesinclude, but are not limited to, cholinesterases (ChE), acetylcholineesterase (AChE) and aromatic L-amino acid decarboxylase (AADC).

Preferred headgroups according to the invention are those which serve assubstrates to enzymes at a target site of a biological environment, e.g.hydrolytic enzymes, enhance transport of the nanoparticles throughbiological barriers and/or stabilize a vesicular structure of thenanoparticles. At least one of these preferred headgroups is aselectively cleavable headgroup.

Non-limiting examples of such headgroups include: (i) choline orthiocholine, O-alkyl, N-alkyl or ester derivatives thereof. O-alkylderivatives of choline or thiocholine are derivatives in which the Hatom of the hydroxy group is replaced with a straight or branched C1-C20alkyl and include methyl, ethyl, propyl, butyryl, pentyl, hexyl andoctyl choline/thiocholine. N-alkyl derivatives of choline andthiocholine are derivatives in which one, two or three of the methylgroups attached to the quaternary nitrogen atom are replaced by astraight or branched C1-C20 alkyl. Choline and thiocholine estersinclude, for example, acetylcholine, acetylthiocholine, propionylcholine/thiocholine, butanoyl choline/thiocholine, pentanoylcholine/thiocholine, hexanoyl choline/thiocholine, octyanoylcholine/thiocholine. Such choline and thiocholine derivatives may becleaved by choline or acetylcholine esterases found in the brain; (ii)non-aromatic amino acids with functional side chains such as glutamicacid, aspartic acids, lysine or cysteine, or an aromatic amino acid suchas tyrosine, tryptophan, phenylalanine and derivatives thereof such aslevodopa (3,4-dihydroxy-phenylalanine) and p-aminophenylalanine. Thecarboxyl group of the aromatic amino acids is selectively cleaved byaromatic AADCs found in brain cells; (iii) a peptide or a peptidederivative that is specifically cleaved by an enzyme at a diseased site.Non-limiting examples include enkephalin which is cleaved byenkephalinase primarily in the brain; N-acetyl-ala-ala, which is cleavedby elastase that is overexpressed in certain types of cancer andaneurysms; a peptide that constitutes a domain recognized by beta andgamma secretases (which are over expressed in the brain of Alzheimer'sdisease patients), or a peptide that is recognized by stromelysins.Nanoparticles comprising these peptides will release their content ininflammatory sites; (iv) saccharides such as glucose, mannose andascorbic acid; (v) other compounds such as nicotine, cytosine, lobeline,polyethylene glycol, or cannabinoids.

In certain embodiments, one or more of the headgroups of themulti-headed amphiphiles are amphoteric and have a pI point. Examples ofsuch headgroups are amino acids, which may have a net anionic charge ata pH above their pI point or a net cationic charge at a pH below theirpI point. The pH during the formation of the nanoparticles can beadjusted such that the headgroups on the amphiphiles will have anopposite charge to that of the active agent. In some cases, the pH maybe changed after nanoparticle formation in order to facilitatecomplexation with the active agent.

In certain embodiments, the active agents e.g., proteins and peptidesmay have both fixed anionic and cationic groups or amphoteric groups,and complexation with charged headgroups of the amphiphiles isfacilitated as a function of the pH in which the nanoparticles areformed: at a pH below the pI point of the peptides or proteins, they mayhave a net cationic charge and can form complexes with amphiphileshaving a net anionic charge at this pH, whereas at a pH above the pIpoint of the peptides or proteins they may have a net anionic charge andform complexes with amphiphiles having a net cationic charge.

In certain embodiments, the active agent molecules which bear chargedgroups are encapsulated within the vesicular core at a certain pH wherethey cannot complex with the headgroups of the amphiphiles since theyboth have the same ionic charge. After vesicle formation andencapsulation, the pH is changed such that residual non-encapsulatedactive agent changes its net charge and forms ionic complexes with theheadgroups. Such complexation strengthen the nanoparticle structure.

In certain embodiments, when the net charges of the active agent and theamphiphiles are opposite, the active agent is predominantly complexedwith ionic groups on the inner and outer surface of the vesicles while aminor amount is within the core. In certain additional embodiments, whenthe headgroups of the amphiphiles are partially or all saturated withactive agent molecules or with non-active additives that are added tothe nanoparticle, a larger fraction of the active molecules is alsoencapsulated within the vesicle core. Non-active additives, which areadded mostly in order to enhance stability, can form counter saltmoieties to the charged headgroups. An example of such an additive ischolesterol hemmisuccinate in which the hemmisuccinate forms a countersalt moiety to cationic headgroups such as acetyl choline.

In certain embodiments, the biological active agent may facilitatestable vesicles structures by being, at least in part, incorporated orembedded into the membrane of the vesicles. In these embodiments, theactive agent has amphiphilic properties and is a molecule comprising anionic group(s) and a predominant hydrophobic structure.

The structure of the nano-sized particle of the inventions is determinednot only by the chemical composition of its components but also by thechemical conditions in which it was formed such as ionic strength, pH,buffers and concentrations of the various components. The nanoparticlestructure may also be a function of the method by which it was prepared,which for example can be film hydration followed by sonication (FHS) orfilm hydration followed by extrusion (FHE), or solvent (e.g., ethanol)injection, optionally followed by sonication and/or extrusion.

Substantial properties of the nanoparticles of the invention include:(i) small, stable size of less than 200 nm, preferably less than 100 nmdiameter, mainly due to optimized packing of the amphiphile components;(ii) protection of the encapsulated material from enzymatic andotherwise chemical modifications; (iii) good blood circulatory life timein order to reach target sites; (iv) penetration through biologicalbarriers; and (v) a selective disruption mechanism at the target site ororgan.

Nano-sized particles encompassed by the present invention may haveconfigurations or aggregate structures other than spherical vesicularcomplexes. For example, a peptide, protein or polynucleotide may besurrounded by a sheet of amphiphiles, preferably bolaamphiphiles, suchthat headgroups of the bolaamphiphiles and counter charged groups on thepeptide interact. This arrangement can change thehydrophobic/hydrophilic structure of a biologically active peptide orprotein.

A spherical or a particle approaching a spherical shape is a preferredshape for targeted release application of active molecules, particularlyfor injectable formulations and oral dosage forms, which enter the bloodcirculatory system. Other configurations have other applications such asimplants of anti cancer drugs at tumor sites for slow controlled releaseor anti microbial activity in organs like the heart or lungs.

Bolaamphiphiles are the preferred amphiphiles for the purpose of thepresent invention, particularly since they form monolayer vesicles.Monolayer vesicles are advantageous since they are far more stable in abiological system due to substantially reduced lipid exchange with thecell membrane, as compared to bilayer and multilayer vesicles andliposomes, let alone vesicles and liposomes comprising phospholipids.Lipid exchange is crucial for intact penetration through biologicalbarriers and increased blood circulatory lifetime. Minimal lipidexchange of the vesicular membrane with cellular membrane increasesstability.

Though highly stable structures, monolayer vesicles made frombolaamphiphiles bearing the proper selectively cleavable headgroups cannevertheless be disrupted at a given site, which contains enzymes insufficient concentrations for facilitating hydrolysis. Selectivedisruption mechanisms are more easily obtained with monolayer membranescompared to bilayer membranes as bolaamphiphiles readily change theirself aggregate structures upon relative small changes in their molecularstructures. Thus, removing the headgroups on monolayer vesicles madefrom bolaamphiphiles may more readily disrupt the vesicular structureand release the encapsulated material at the site of hydrolysis.

In particular embodiments of the invention at least one hydrogen-bondinggroup such as, but not limited to, —OH, —SH, —NH—, —N⁺H₂—, —NH₂, —N⁺H₃,—NH—CO—, —O—CO—NH—, —NH—CO—NH—, —C═NOH, —C(NH₂)═NOH, —C(NH₂)═NO— and—CO—NH₂, is found either within the selectively cleavable headgroup orwithin the headgroup containing the selectively cleavable group ormoiety and/or in close proximity thereto, thus imparting more stabilityand other features to the vesicles made from such amphiphilic compounds.By “close proximity” it is meant herein that the hydrogen-bonding groupis located at the atom vicinal to the atom of the aliphatic chain towhich the headgroup is bound and/or at a distance of up to 6 atoms inthe aliphatic chain. Bolaamphiphiles comprising the aforementionedhydrogen bonding groups suitable for the purpose of the invention arethose disclosed in WO 30/0474499 incorporated herein by reference as iffully disclosed herein.

In particular embodiments, the nanoparticles of the invention compriseat least one bolaamphiphile compounds having the formula I:

X₁—CO—X₂—X₃  [I]

wherein

X₁ is -Q₁-R₀, wherein Q₁ is —NH—, —O—, —S—, or —O—PO(OH)—O—;

R₀ is —X₄—X₅—X₆ or —(CH₂)r′-X₇;

X₂ and X₅, the same or different, each is an alkylene chain of at least5 carbon atoms;

X₃ and X₆, the same or different, each is an aliphatic chain of at least5 and at most 18 carbon atoms optionally carrying at least one doublebond, said aliphatic chain being substituted by at least one polar,ionic and/or epoxy groups and/or by at least one moiety containing atleast one polar, ionic and/or epoxy groups, said at least one polar,ionic and/or epoxy groups and at least one moiety containing at leastone polar, ionic and/or epoxy groups being, in relation to theirsubstitutions, in any combination of 1-2, 1-2-3, 1-2-3-4, 1-2-4-5,1-2-3-4-5, 1-2-4, 1-2-5, 1-3-4, 1-3, 1-5, 1-4, or 1-2-6 positions of thechain, the position 1 being arbitrarily assigned to the substitutionmost remote from the CO group;

X₄ is a spacer group consisting of a linear or branched aliphatic chainof up to 16 carbon atoms, optionally interrupted by Q₂ or by —CO-Q₂-,wherein Q₂ is —NH—, —O—, —S—, or —O—PO(OH)—O—, and optionally containingat least one polar and/or ionic group or at least one moiety containingat least one polar and/or ionic group;

X₇ is hydrogen, C₆-C₁₄ aryl, preferably phenyl, or a heterocyclicradical;

r′ is an integer from o to 12; and

wherein at least one polar and/or ionic group and/or at least one moietycontaining at least one polar and/or ionic group of X₃, X₄ and/or X₆ isa headgroup, and wherein at least one of said headgroup is a selectivelycleavable headgroup or a headgroup containing a selectively cleavablegroup or moiety and, optionally, at least one hydrogen-bonding group islocated within and/or in close proximity to said selectively cleavableheadgroup or headgroup containing a selectively cleavable group ormoiety.

In other particular embodiments, the nanoparticles of the inventioncomprise amphiphilic compounds having the formula II:

X6-X5-X4-CO-Q1-X2-X3  [II]

wherein

Q₁ is —NH—, —N(CH₃)_(1 to 2)—, —O—, —S—, or —O—PO(OH)—O—;

X₄ is a spacer group consisting of a linear or branched aliphatic chainof up to 16 atoms, optionally interrupted by —CO-Q₂-, wherein Q₂ is—NH—, —N(CH₃)_(1 to 2)—, —O—, —S—, or —O—PO(OH)—O—, and optionallycontaining at least one polar and/or ionic group or at least one moietycontaining at least one polar and/or ionic group;

X₂ and X₅, the same or different, each is an alkylene chain of at least5 carbon atoms;

X₃ and X₆, the same or different, each is an aliphatic chain of at least5 and at most 18 carbon atoms optionally carrying at least one doublebond, said aliphatic chain being substituted by at least one polar,ionic and/or epoxy groups and/or by at least one moiety containing atleast one polar, ionic and/or epoxy groups, said at least one polar,ionic and/or epoxy groups and at least one moiety containing at leastone polar, ionic and/or epoxy groups being, in relation to theirsubstitutions, in any combination of 1-2, 1-2-3, 1-2-3-4, 1-2-4-5,1-2-3-4-5, 1-2-4, 1-2-5, 1-3-4, 1-3, 1-5, 1-4, or 1-2-6 positions of thechain, the position 1 being arbitrarily assigned to the substitutionmost remote from the CO group; and

wherein at least one polar and/or ionic group and/or at least one moietycontaining at least one polar and/or ionic group of X3, X4 and/or X6 isa headgroup, and wherein at least one of said headgroup is a selectivelycleavable headgroup or a headgroup containing a selectively cleavablegroup or moiety and, optionally, at least one hydrogen-bonding group islocated within and/or in close proximity to said selectively cleavableheadgroup or headgroup containing a selectively cleavable group ormoiety.

In one embodiment, the bolaamphiphiles of the formula I or II arecomposed of two fatty acid chains, formed by the —X₂—X₃ and —X₅—X₆groups, each comprising a selectively cleavable polar or ionic headgroupor a polar or ionic headgroup containing a selectively cleavable groupor moiety. In certain embodiments, at least one of the fatty acid chainscontains a ionic or polar hydrogen-bonding group in close proximity tosaid headgroup and/or attached to a site within said headgroup. The twofatty acid chains are separated by a non-fatty acid midsection orspacer, for example a C₂-C₁₆ alkylene chain optionally interrupted by—O—, —S— or —NH—, and each fatty acid chain is bound to the midsectionthrough an amide (a hydrogen-bonding group), ether, ester, thioester,and/or phosphoester bond.

In certain embodiments, said two fatty acid chains may be derived fromthe same or different fatty acids, selected from, but not limited to,vernolic acid (12,13-epoxyoctadec-9-enoic acid), lesquerolic acid(14-hydroxyeicosa-11-enoic acid), ricinoleic acid(12-hydroxyoctadec-9-enoic acid), partially or totally epoxidizedlinoleic, linolenic, and arachidonic acid, or from a derivative thereofobtained by reaction of the epoxy group and/or of a double bond and/orof a hydroxy group, or the aforementioned fatty chains may also bederived from a fatty acid selected from lauric, myristic, palmitic,stearic, arachidic, beherric, lignoceric, or undecylenic acid or from aderivative thereof. The sources of some epoxidized and hydroxylatedfatty acids are vernonia oil, lesquerella oil, castor oil, andepoxidized soya and linseed oil.

In a particular embodiment, the nanoparticles of the invention compriseat least one bolaamphiphile of the formula Ia:

R₈—R₇-A₄-R₆-A₃-R₅—OC-Q₂-R₁₀-Q₁-CO—R₁-A₁-R₂-A₂-R₃—R₄  (Ia)

wherein:

R₁ and R₅, the same or different, each is —(CH₂)_(n);

A₁ is selected from —(CH₂)_(m+2)—, —CH—(CH₂)_(m)—, —CH—CH—CH(Y₁)—,—CH₂—CH₂—CH(Y₁)—, —CH₂—CH(Y₁)—(CH₂)_(m)—, —CH(Y₁)—CH₂—(CH₂)_(m)—,—CH(Y₁)—CH(Y₂)—(CH₂)_(m)—, wherein Y₁ and Y₂ each is halogen, —OH,—O—CO—(CH₂)_(m)—Y₃, —NH—CO—Y₃, —SH, —SR₁₁, —NH₂, or —N(R₁₁)(R₁₂), or Y₁and Y₂ together with the carbon atoms to which they are attached form a2,3-oxiranylene group; and Y₃ is halogen, —OH, —SH, —NH₂, or—N(R₁₁)(R₁₂);

R₂ and R₆, the same or different, each is C1-C4 alkylene, preferablymethylene, optionally substituted by halogen, amino or hydroxy;

A₂ is selected from —CH(R₁₃)—, —CH₂—CH(R₁₃)—, —CH(R₁₃)—CH₂—,—CH(OH)—CH(R₁₃)—, —CH(R₁₃)—CH(OH)—, —CH(OH)—CH₂—CH(OH)—CH(R₁₃)—,—CH(OH)—CH₂—CH(R₁₃)—CH(OH)—, -G1-(C6-C14 arylene)-(CH₂)_(q)R₁₄,—N(CH₃)₂R₁₄, or —SR₁₄;

R₃ and R₇, the same or different, each is —(CH₂)_(o)—;

R₄ is H or CH₃, and wherein the total sum of carbon atoms in theR₁-A₁-R₂-A₂-R₃—R₄ chain is at most 23;

Q₁ is —NH—, —O—, —S—, or —O—PO(OH)—O—;

Q₂ is —NH—, —O—, —S—, or —O—PO(OH)—O—;

R₁₀ is a group selected from —(CH₂)_(p)—; —CH₂(CH₃)—(CH₂)_(p)—;—CH(CH₃)—(CH₂)_(p)—CH(CH₃)—; —(CH₂—CH₂—O—)_(p)—CH₂—CH₂—;—(CH₂—CH₂—S—)_(p)—CH₂—CH₂—; —(CH₂—CH₂—NH—)_(p)—CH₂—CH₂—; —C6-C14arylene-; —(C6-C14 arylene)-R—(C6-C14 arylene)-, wherein R is C1-C4alkylene, —C(CH₃)₂—, —O—, —S—, —NH— or —SO₂—;

A₃ is as defined for A₁, or is —(CH₂)_(m), phenyl or —CH₂-phenyl,wherein the phenyl ring may be substituted by C1-C4 alkyl and/or byhalogen;

A₄ is as defined for A₂, or is —(CH₂)_(m);

R₈ is as defined for R₄;

R₁₁ and R₁₂, the same or different, each is C1-C18 alkyl optionallysubstituted by halogen; phenyl or —CH₂-phenyl, wherein the phenyl ringmay be substituted by C1-C4-alkyl and/or by halogen, and wherein one ofR₁₁ and R₁₂ may be H;

R₁₃ is -G1-(CH₂)_(m)R₁₄ or -G1-CO(CH₂)_(m)R₁₄;

G1 is —O—, —S—, —NR″—, —CH₂NR″—, —CH₂S— or —CH₂O—, —NH—CO—, —O—CO—NH—,—NH—CO—NH—, —C═NO—, —C(NH₂)═NO—, wherein R″ is H or C1-C18 alkyl;

R₁₄ is either a selectively cleavable head group or a head groupcontaining a selectively cleavable group or moiety, or is as defined forR₁₅ or for R₁₅ substituted by a selectively cleavable group or moiety;

R₁₅ is —NH₂; —NR₁₁R₁₂; —N⁺R₁₁R₁₂R₁₆ wherein R₁₆ is as defined for R₁₁and R₁₂; —O—CO—(C2-C6 alkenyl); —O—CO—(CH₂)_(t)—NR₁₁R₁₂;—O—CO—(CH₂)_(t)—N⁺R₁₁R₁₂R₁₆; —O—CO—(CH₂)_(t)—COOH; —O—CO—(CH₂)_(t)—SO₃H;—O—CO—(CH₂)_(t)—O—PO(OH)₂; —NH—(CH₂)_(r)—COOH; —NH—(CH₂)_(r)—SO₃H;—NH—(CH₂)_(r)—O—PO(OH)₂; —NH—PO(OH)₂; —N⁺(CH₃)₂—R₁₇;—O—PO(OH)—O—(CH₂)₂—N⁺R₁₁R₁₂R₁₆; —O—PO(OH)—O—(CH₂)₂—NH₃ ⁺;—O—PO(OH)—NH—PO(OH)—O—; —O—PO(OH)—O—CH₂—CH(N⁺H₃)—COO⁻; —CH₂—CH═CH₂;—CO—CH═CH₂; —CO—C(CH₃)═CH₂; —(CH₂)_(r)—COOH; —(CH₂)_(r)—O—SO₂H;—(CH₂)_(r)—O—PO(OH)₂; —SR₁₈; -G1-(C6-C14 arylene)-NR₁₁R₁₂; -G1-(C6-C14arylene)-N⁺R₁₁R₁₂R₁₆; -G1-(C6-C14 arylene)-COOH; -G1-(C6-C14arylene)-SO₃H; -G1-(C6-C14 arylene)-O—PO(OH)₂; -G1-(C6-C14arylene)-(CH₂)_(t)—NR₁₁R₁₂; -G1-(C6-C14 arylene)-(CH₂)_(n)—N⁺R₁₁R₁₂R₁₆;-G1-(C6-C14 arylene)-(CH₂)_(t)—COOH; -G1-(C6-C14arylene)-(CH₂)_(t)—SO₃H;

R₁₇ is —CH₂—CH═CH₂, —CO—CH═CH₂, —CO—C(CH₃)═CH₂, —(CH₂)_(q)—N⁺R₁₁R₁₂R₁₆,—(CH₂)_(q)—NH—(CH₂)_(q)—SO₃H, —(CH₂)_(q)—NH—(CH₂)_(q)—COOH,—(CH₂)_(q)—NH—(CH₂)_(q)—O—PO(OH)₂, —PO(OH)₂, or—O—PO(OH)—O—(CH₂)₂—N⁺R₁₁R₁₂R₁₆;

R₁₈ is hydrogen, C1-C18 alkyl, C2-C6 alkenyl with a terminal doublebond, —CO—CH═CH₂, or —CO—C(CH₃)═CH—NR₁₁R₁₂;

n is an integer from 5 to 10; m is an integer from 0 to 4; o is aninteger from 0 to 10; p is an integer from 1 to 16; q is an integer from0 to 3; r is an integer from 1 to 6; and t is an integer from 1 to 14,

and salts thereof.

In one embodiment, the amphiphilic compound Ia is symmetric andcomprises two identical fatty acid chains, to each of which the sameselectively cleavable headgroup or the same headgroup containing thesame selectively cleavable group or moiety are attached along with thesame stabilizing polar hydrogen-bonding group. The hydrogen-bondinggroup may be either attached to the aliphatic chain in the sameproximity to said headgroup or situated within each of the headgroups.

As used herein the term “C1-C18 alkyl” typically refers to a straight orbranched alkyl radical having 1-18 carbon atoms and includes, forexample, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl,tert-butyl, n-heptyl, 2,2-dimethylpropyl, n-hexyl, n-dodecyl,n-octadecyl and the like. The term “C2-C16 alkylene” refers to straightor branched alkylene groups having 2-16 carbon atoms and includes forexample methylene, ethylene, propylene, butylene and the like. The term“C2-C6 alkenyl” refers to straight or branched hydrocarbon radicalshaving 2-6 carbon atoms and at least one terminal double bond andincludes for example vinyl, prop-2-en-1-yl, but-3-en-1-yl,pent-4-en-1-yl, and hex-5-en-1-yl. The term “aliphatic chain of up to 16atoms optionally interrupted by Q₂ or —CO-Q₂” means that the chainincluding the heteroatoms represented by Q₂ has up to 16 atoms.

The term “C6-C14 aryl” refers to an aromatic carbocyclic group having 6to 14 carbon atoms consisting of a single ring or multiple condensedrings such as phenyl, naphthyl, and phenanthryl optionally substitutedby C1-C6 alkyl. The term “heterocyclic” refers to a monocyclic, bicyclicor tricyclic fused-ring heteroaromatic group. Particular examples arepyridyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, quinolinyl,thiazolyl, pyrazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuryl,isobenzofuryl, indolyl, imidazo[1,2-a]pyridyl, benzimidazolyl,benzthiazolyl and benzoxazolyl. The term “halogen” refers to fluoro,chloro, bromo or iodo.

For the preparation of some of the compounds of formula Ia and IIa,methods similar to those described in WO 03/047499 and WO 02/055011,both of the same applicant, can be used.

Some of the amphiphilic derivatives used for the preparation ofnanoparticles according to the invention are new and their synthesis isdescribed in the examples disclosed herein.

Symmetric amphiphilic compounds of the formula Ib, for example, can besynthesized starting from vernolic acid, which acyl residue has theformula:

by reaction with an alkylene diamine, e.g. ethylene diamine, and openingof the oxiranyl ring with a carboxylic acid derivative, to obtain aderivative Ib:

R₁₉—CO—NH—(CH₂)₂—NH—CO—R₁₉  (Ib)

wherein R₁₉ is —(CH₂)₇—CH═CH—CH₂—CH(OH)—CH(R₂₀)—(CH₂)₄—CH₃

and R₂₀ is —OCOCH₂CH₂NH-phenyl-CH₂—CH(NH₂)—COOH.

In this example, R₂₀ is the headgroup moiety containing the selectivelycleavable moiety p-aminophenylalanine that is linked to the fatty acidchain R₁₉ through an ester linkage, and said headgroup contains thehydrogen-bonding —NH group at the para position of the phenyl group andanother hydrogen-bonding —OH group on the vicinal carbon atom (positions1-2), both contributing to the stabilization of the nanoparticles basedthereon.

Instead of ethylene as the spacer X4, another longer linear spacer or,for example, a branched spacer can be formed by reaction with a diaminesuch as: NH₂—CH₂—CH(CH₃)—CH₂—CH(CH₃)—CH₂—NH₂.

In another embodiment, the two fatty acid chains can be linked to thespacer by an ester instead of amide linkages, when the reaction isconducted with a dihydroxy compound such as diethylene glycol, thusobtaining, for example a compound of formula Ic:

R₁₉—CO—O—(CH₂)₂—O—CO—R₁₉  (Ic)

wherein R₁₉ is as defined for compound Ib above.

In a further embodiment, the bolaamphiphile has the formula Id:

R′₁₉—CO—NH—(CH₂)₂—NH—CO—R′₁₉  (Id)

wherein R′₁₉ is —(CH₂)₇—CH═CH—CH₂—CH(OH)—CH(R₂₁)—(CH₂)₄—CH₃;

and R₂₁ is —NHCO—CH₂CH₂NH-phenyl-CH₂—CH(NH₂)—COOH.

In a still further embodiment, the amphiphilic compound has the formulaIe:

R′₁₉—CO—NH—(CH₂)₂—NH—CO—R″₁₉  (Ie)

wherein R′₁₉ is as defined above, R″₁₉ is—(CH₂)₇—CH═CH—CH₂—CH(OH)—CH(R₂₃)—(CH₂)₄—CH₃ and R₂₃ is—NH—CH₂CH₂NH-phenyl-CH₂—CH(NH₂)—COOH.

In the compounds of the formulas Id and Ie, the headgroup moiety R₂₁ orR₂₃ containing the p-aminophenylalanine group is linked to the fattyacid chain R′₁₉ or R″₁₉ through an amido or amino linkage, respectively,and said headgroup contains the hydrogen-bonding —NH group at the paraposition of the phenyl group and the hydrogen-bonding —CONH— or —NH—group two carbon atoms further, besides the hydrogen-bonding —OH groupon the vicinal carbon atom. These compounds can be obtained fromvernonia oil by amidation (Id) or aminolysis of the epoxide ring (Ie).

In certain embodiments of the present invention, nanoparticles areprovided containing asymmetric bolaamphiphiles in which the aliphaticchains on both sides of the spacer X4 are identical, except for theheadgroups, and wherein at least the bulkier headgroup contains theselectively cleavable group or moiety and will be on the outside of thenanoparticle surface. An example of such an asymmetric compound is acompound of formula If:

R₂₄—CO—NH—(CH₂)₂—NH—CO—R′₁₉  (If)

wherein R″₁₉ is as defined above, and contains the bulkier headgroup R₂₁with the selectively cleavable moiety—NH—CH₂CH₂NH-phenyl-CH₂—CH(NH₂)—COOH;

R₂₄ is —(CH₂)₇—CH═CH—CH₂—CH(OH)—CH(R₂₅)—(CH₂)₄—CH₃, and

R₂₅ is —NH—CO—CH₂—COOH, a headgroup with no selectively cleavable group.

In still another embodiment, the nanoparticles comprise the symmetricbolaamphiphile of the formula Ig:

R₂₇—CO—NH—(CH₂)₂—NH—CO—R₂₆  (Ig)

wherein

R₂₆ is —(CH₂)₁₂—CH(OH)—CH₂—R₂₃

R₂₇ is —(CH₂)₁₂—CH(OH)—CH₂—R₂₃, and

R₂₃ is —NHCH₂CH₂NH-phenyl-CH₂—CH(NH₂)COOH.

The compound Ig is a compound of formula Ia wherein R₁ and R₅ are—(CH₂)₁₀, A₁ and A₃ are —CH₂—CH₂—CH(Y₁)—, Y₁ is —OH, R₂ and R₆ are —CH₂,A₂ and A₄ are R₂₃, R₃ and R₇ are absent and R₄ and R₈ are H.

Compounds Ih-In below are some specific examples of amphiphiliccompounds in which a hydrogen-bonding group is located within theheadgroup containing the selectively cleavable group or moiety.

R₂₈—CO—NH—(CH₂)₂—NH—CO—R₂₈  (Ih)

wherein R₂₈ is —(CH₂)₁₂—R₂₃, and R₂₃ is—NHCH₂CH₂NH-phenyl-CH₂—CH(NH₂)COOH

Compound Ih has the headgroup R₂₃ that contains both the selectivelycleavable phenylalanine (levodopa-type) moiety and the hydrogen-bonding—NH— group attached to a (CH₂)₁₂ aliphatic chain with no furtherhydrogen-bonding groups in the aliphatic chain.

R₂₉—CO—NH—(CH₂)₂—NH—CO—R₂₉  (Ii)

wherein R₂₉ is —(CH₂)₁₂—R₂₁, and R₂₁ is—NHCO—CH₂CH₂NH-phenyl-CH₂—CH(NH₂)COOH.

The sole difference between the compounds Ih and Ii is that theheadgroup moiety R₂₃ in Ih is attached to the (CH₂)₁₂ aliphatic chain byan amino linkage while the headgroup moiety R₂₃ is attached by an amidolinkage.

Compound Ij is an example of an asymmetrical amphiphilic compound with abulky headgroup R₂₉ containing the levodopa-type headgroup on one endand a smaller headgroup with a —COOH group on the other end:

R₃₀—CO—NH—(CH₂)₂—NH—CO—R₂₉  (Ii)

wherein R₂₉ is —(CH₂)₁₂—R₂₁, and R₂₁ is—NHCO—CH₂CH₂NH-phenyl-CH₂—CH(NH₂)COOH, and R₃₀ is —(CH₂)₁₂—COOH.

Another example of a symmetrical amphiphilic compound is represented byformula Ik:

R₃₁—CO—NH—(CH₂)₂—NH—CO—R₃₁  (Ik)

wherein R₃₁ is —(CH₂)₁₂—R₃₂, and R₃₂ is—NHCH₂CH₂N⁺(CH₃)₂—CH₂—CH₂—OCOCH₃.

R₃₂ is an example of a headgroup containing both an acetylcholine-typegroup and the hydrogen-bonding —NH— group.

Another example of an asymmetrical amphiphilic derivative is a compoundof the formula Il:

R₃₃—CO—NH—(CH₂)₂—NH—CO—R₃₁  (Il)

wherein R₃₁ is —(CH₂)₁₂—R₃₂, and R₃₂ is—NHCH₂CH₂N⁺(CH₃)₂—CH₂—CH₂—OCOCH₃, and

R₃₃ is —(CH₂)₁₂—R₃₄, and R₃₄ is —NHCH₂CH₂N⁺(CH₃)₃.

In the asymmetrical bolaamphiphile Il, one chain contains the bulkierionic headgroup R₃₂ with the acetylcholine-type group and thehydrogen-bonding —NH— group, while the other aliphatic chain containsthe smaller ionic headgroup.

Similarly to the amphiphilic compounds derived from vernolic acid andfrom saturated fatty acids exemplified above, further amphiphilicderivatives are derived from the lesquerolic acid found in lesquerellaoil, which acyl residue has the formula:

—CO—(CH₂)₉—CH═CH—CH₂—CH(OH)—(CH₂)₅CH₃

In one embodiment, a symmetric bolaamphiphile has the formula Im:

R₃₅—CO—NH—(CH₂)₂—NH—CO—R₃₅  (Im)

wherein R₃₅ is —(CH₂)₉—CH═CH—CH₂—CH(R₃₆)—(CH₂)₅CH₃;

and R₃₆ is —OCH₂CH₂NH—CO—CH₂—CH₂—CH(NH₂)COOH.

R₃₆ is a headgroup containing the selectively cleavable residue ofglutamic acid —CH₂—CH₂—CH(NH₂)COOH, and the hydrogen-bonding —CONH—polar group for stabilization.

In an additional embodiment, the nanoparticles of the invention comprisebolaamphiphiles derived from the ricinoleic acid found in castor oil,which acyl residue has the formula:

—CO—(CH₂)₇—CH═CH—CH₂—CH(OH)—(CH₂)₅CH₃

As an example, a symmetrical derivative has the formula In:

R₃₇—CONH(CH₂)₂NH—CO—R₃₇  (In)

wherein R₃₇ is —(CH₂)₇—CH═CH—CH₂—CH(R₃₈)—(CH₂)₅CH₃, and

-   -   R₃₈ is —OCH₂CH₂NH-phenyl-CH₂—CH(NH₂)COOH.

In a different embodiment of the invention, the nanoparticle comprise anamphiphilic compound that has, besides the polar or ionic cleavableheadgroups or headgroups containing the selectively cleavable groups andoptionally hydrogen-bonding groups, additional hydrophobic pendantseither on the aliphatic chain and/or on the cleavable headgroup orheadgroup containing the selectively cleavable group or moiety.

In one embodiment, a symmetric amphiphilic compound having a hydrophobicpendant on the headgroup containing the selectively cleavable group hasthe formula Io:

R₃₉—CONH(CH₂)₂NH—CO—R₃₉  (Io)

wherein R₃₉ is —(CH₂)₁₂—N(R₄₀)CH₂CH₂NH-phenyl-CH₂—CH(NH₂)COOH;and R₄₀ is a C4-C16 alkyl.

In the above compound, the headgroups that contain the selectivelycleavable levodopa-type group and the H-bonding group —NH—, also have arelatively long aliphatic chain R₄₀ attached to an amino group in bothheadgroup moieties to give extra stability due to hydrophobicinteractions.

In another embodiment, an additional hydrophobic group of theamphiphilic compound (R₄₀) is not located in the headgroup moiety asabove, but is bound to the fatty acid chain through an ether linkage, asshown in formula Ip:

R₄₁—CONH(CH₂)₂NH—CO—R₄₁  (Ip)

wherein

R₄₁ is —(CH₂)₇—CH═CH—CH₂—CH(OR₄₀)—CH(R₃₈)—(CH₂)₄CH₃

R₄₀ is C4-C16 alkyl, and

R₃₈ is —OCH₂CH₂NH-phenyl-CH₂—CH(NH₂)COOH.

In particular embodiments, the nanoparticles of the invention comprise abolaamphiphile of the formula IIa comprising a —CO—R₁₀—CO— midsection asfollows:

R₈—R₇-A₄-R₆-A₃-R₅-Q₂-CO—R₁₀—CO-Q₁-R₁-A₁-R₂-A₂-R₃—R₄  (IIa)

wherein all groups are as defined hereinabove and Q₂ and Q₁ areidentical.

Such bolaamphiphiles can be synthesized starting, for example, fromhexadecanoic acid [HO₂C(CH₂)₁₄CO₂H] and 11-hexadecen-1-ol[CH₃(CH₂)₃CH═CH(CH₂)₁₀OH], to obtain the following symmetric derivativesIIb and IIc with a headgroup containing a glutamic acid residue or ap-aminophenylalanine residue, respectively:

R₄₂—O—CO—(CH₂)₁₄—CO—O—R₄₂

wherein R₄₂ is —(CH₂)₁₀—CH(OH)—CH(R₄₃)—(CH₂)₃—CH₃, and

R₄₃ is —O—CO—CH₂CH₂CH(NH₂)CO₂H  (IIb)

or R₄₃ is R₂₁: —NH—CO—CH₂CH₂CH—NH-phenylalanine  (IIc)

An asymmetric bolaamphiphile can be made having one bulkier headgroupcontaining a p-aminophenylalanine residue and a second smaller headgroupcontaining a glutaric acid residue, as shown by formula IId:

R₄₄—O—CO—(CH₂)₁₄—CO—O—R₄₅  (IId)

wherein

R₄₄ is —(CH₂)₁₀—CH(OH)—CH(R₄₆)—(CH₂)₃—CH₃;

R₄₅ is —(CH₂)₁₀—CH(OH)—CH(R₄₇)—(CH₂)₃—CH₃;

R₄₆ is —NHCO—CH₂CH₂CH—NH-phenyl alanine;

and R₄₇ is —NHCO—CH₂CH₂CH₂CO₂H

In particular embodiments, a symmetric bolaamphiphiles do not havehydrogen-bonding —OH group on the vicinal carbon atom as in compoundsIIb-IId above, but rather the hydrogen-bonding group is located withinthe headgroup containing an ionic selectively cleavable group, as shownin formula IIe:

R₄₈—O—CO—(CH₂)₁₆—CO—O—R₄₈  (IIe)

wherein R₄₈ is —(CH₂)₁₁—NH—(CH₂)—N⁺(CH₃)₂(CH₂)₂O—CO—CH₃Cl⁻;

This symmetric bolaamphiphile has a headgroup R₄₈ containing both theacetylcholine-type moiety and the hydrogen-bonding —NH— group, and canbe prepared starting from HOOC—(CH₂)₁₆—COOH and 11-bromo-1-undecanol[Br(CH₂)₁₁OH].

In a further embodiment, starting again from HOOC—(CH₂)₁₆—COOH and11-bromo-1-undecanol, the following asymmetric bolaamphiphile of formulaIIf can be made with one acetylcholine headgroup and one glucosamineheadgroup:

R₄₉—O—CO—(CH₂)₁₆—CO—O—R₅₀  (IIf)

wherein R₄₉ is —(CH₂)₁₁—NH—(CH₂)—N⁺(CH₃)₂(CH₂)₂O—CO—CH₃Cl⁻;

and R₅₀ is —(CH₂)₁₁—NH—(CH₂)—NH—C₆H₁₁O₅

wherein —NH—C₆H₁₁O₅ is the glucosamine moiety, useful for transportacross the biological barriers.

In particular embodiments, the nanoparticles of the invention compriseone or more bolaamphiphiles of the formulas IIa, comprising a—CO—R_(n)—CO— midsection, wherein n is 2-12, and a hydrogen-bondinggroup —OH located adjacent to the selectively cleavable headgroup on thevicinal carbon atom (positions 1-2). In more particular embodiments thebolaamphiphiles are the symmetric bolaamphiphiles herein designatedDerivative 1, Derivative 4 and Derivative 5, or the asymmetricbolaamphiphile herein designated Derivative 3.

Derivative 4 is a known compound extensively used for preparation ofnanoparticles according to the present invention. The symmetricDerivative 1 and Derivative 4 comprise the same selectively cleavableheadgroup acetylcholine. In Derivative 1 the acetylcholine is linkedthrough the oxygen atom of the acetyl moiety, whereas in Derivative 4the acetylcholine is linked through one of the N⁺-methyl groups. The wayby which acetylcholine is bound to the fatty chain influences theability of AChE to hydrolyze the headgroup and determines the selectivedisruption mechanism. When the choline ester is bound to the alkyl chainvia the N+-methyl, AChE hydrolyzes the head group and decapsulates theactive agent. However, when the choline ester head group is attached viathe oxygen atom of the acetyl moiety, AChE will not as rapidly hydrolyzethe head group and release of the encapsulated active agent will besubstantially delayed.

In particular embodiments, the nanoparticles comprise one or moreasymmetric bolaamphiphiles of the formula Ia. In a more particularembodiment the asymmetric bolaamphiphile is the compound hereindesignated Derivative 2, which comprises a terminal acetylcholine headgroup and a non-terminal acetylcholine headgroup located adjacent to anOH group (positions 1-2).

In certain embodiment, the nanoparticles comprise bolaamphiphiles whichdo not have a hydrogen-bonding group located adjacent to the cleavableheadgroup. Such bolaamphiphiles may be prepared from ricinoleic acidbased on castor oil. In particular embodiments, these bolaamphiphilesare symmetric bolaamphiphiles of the formula IIa. In more particularembodiments, these bolaamphiphiles are selected from the compoundsherein designated Derivative 6, Derivative 7 and Derivative 8.

In further particular embodiments, the bolaamphiphiles are asymmetricbolaamphiphiles of the formula IIa. In more particular embodiments,these bolaamphiphiles are selected from the compounds herein designatedDerivative 9 and Derivative 10.

The invention further encompasses the salts of the aforementionedbolaamphiphiles. Examples of salts include, but are not limited to acidaddition salts formed with inorganic acids (hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike) and salts formed with organic acids such as acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, maleicacid, ascorbic acid, benzoic acid, and the like. Said compounds can alsobe quaternary salts known by a person skilled in the art, whichspecifically include the quaternary salt of the formula —NRR′R″+Z′wherein R, R′, R″ is independently hydrogen, alkyl or benzyl and Z is acounterion, including chloride, bromide, iodide, O-alkyl,toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate.

Base addition salts are formed with metals or amines such as alkali andalkaline earth metals or organic amines. Examples of metals used ascations are sodium, potassium, magnesium, calcium, and the like.Examples of suitable amines are N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, andN-methylglucamine.

In certain embodiments, the nanoparticles of the present inventioncomprise additives which themselves are amphiphilic derivatives.

In a particular embodiment, the amphiphilic additive has one headgroupand is capable of forming bilayered vesicles. In a more particularembodiment, this amphiphile comprises two fatty acid chains linked to amidsection/spacer region such as —NH—(CH₂)₂—N—(CH₂)₂—N—, or—O—(CH₂)₂—N—(CH₂)₂—O—, and a sole headgroup preferably a selectivelycleavable headgroup or one containing a polar or ionic selectivelycleavable group or moiety, which is localized in said midsectionpreferably attached to the central N atom in the middle of themidsection region. The midsection also contains hydrogen-bonding groupsprovided by the —CONH— groups or polar ester groups, —C(═O)O— at theintersection with the fatty acid chains, and the headgroup moiety mayalso contain hydrogen-bonding groups.

These amphiphilic additives are added to the bolaamphiphile reactionmixture in the process of making nanoparticles in a controlled amount toenable the formation of monolayer vesicles by the predominantbolaamphiphiles. Some examples for different single head amphiphileswith cleavable headgroups are given in WO 03/047499 of the sameinventors.

Thus, bolaamphiphiles comprising selectively cleavable headgroupsbearing net ionic charge, preferably cationic headgroups, areadvantageous according to the invention for three main reasons: (a) theyform vesicles with good circulatory survival and ability of penetrating,intact, through biological barriers such as the blood-brain barrier(BBB), for example via transcytosis; (b) they provide vesicles with aselective disruption mechanism in the CNS, which enables the release ofthe encapsulated material in the brain in a controlled manner; and (c)they exhibit excellent encapsulation efficiency with anionic moleculessuch as peptides, proteins and nucleotides, via both complexation andencapsulation.

Bolaamphiphiles can be classified as either symmetrical or asymmetricalwith respect to their headgroups. In symmetric bolaamphiphiles, bothheadgroups are the same whereas in asymmetrical bolaamphiphiles theheadgroups are different with respect to their structure, electriccharge and/or bulkiness.

In certain embodiments, the bolaamphiphiles are asymmetricbolaamphiphiles that have two headgroups of different size or bulkiness.The difference in headgroups size may vary from very small,intermediate, to a very large difference, and can be optimized tomaximize amphiphile packing and thus vesicle stability for abolaamphiphile with a given total size and a given span between the twoheadgroups on the aliphatic chain. Optimized size calculations forbolaamphiphiles with different headgroups are well known in the art.

Bolaamphiphiles may also be asymmetric with respect to their aliphatictails or with respect to the nature and/or location of polar or hydrogenbonding groups on the aliphatic chain, relative to the headgroups.Additional asymmetry of bolaamphiphiles may be attributed to differencesin aliphatic chain cross-sectional areas, and the presence or absence ofaliphatic or aromatic chain pendants.

Asymmetric bolaamphiphiles may more readily form stable vesicles. Insmall vesicles formed from bolaamphiphiles bearing headgroups ofdifferent sizes, the size-difference can accommodate the differences inradii of curvature on the inner and outer surfaces of the vesicles, withthe larger headgroups presented on the outer surface to maximizeamphiphilic packing and intermolecular interactions.

Symmetrical bolaamphiphiles can form relatively stable structures by theuse of additives such as cholesterol and cholesterol derivatives (e.g.,cholesterol hemisuccinate, cholesterol oleyl ether, anionic and cationicderivatives of cholesterol and the like), or other additives includingsingle headed amphiphiles with one, two or multiple aliphatic chainssuch as phospholipids, zwitterionic, acidic, or cationic lipids.Examples of zwitterionic lipids are phosphatidylcholines,phosphatidylethanol amines and sphingomyelins. Examples of acidicamphiphilic lipids are phosphatidylglycerols, phosphatidylserines,phosphatidylinositols, and phosphatidic acids. Examples of cationicamphipathic lipids are diacyl trimethylammonium propanes, diacyldimethylammonium propanes, and stearylamines cationic amphiphiles suchas spermine cholesterol carbamates, and the like, in optimumconcentrations which fill in the larger spaces on the outer surfaces,and/or add additional hydrophilicity to the particles. Such additivesmay be added to the reaction mixture during formation of nanoparticlesto enhance stability of the nanoparticles by filling in the void volumesof in the upper surface of the vesicle membranes.

Proper design of bolaamphiphiles for a stable delivery system accordingto the invention will usually include long aliphatic segments spanningbetween their headgroups, one or more aliphatic chain pendants andasymmetry in head group size.

In certain embodiments, additives which may be bolaamphiphiles or singleheaded amphiphiles, comprise one or more branching alkyl chains bearingpolar or ionic pendants, wherein the aliphatic portions act as anchorsinto the vesicle's membrane and the pendants (e.g., chitosan derivativesor polyamines or certain peptides) decorate the surface of the vesicleto enhance penetration through various biological barriers such as theintestinal tract and the BBB, and in some instances are also selectivelyhydrolyzed at a given site or within a given organ. The concentration ofthese additives is readily adjusted according to experimentaldetermination.

In certain embodiments, the nanoparticles of the invention comprisemixtures of different multi-headed amphiphiles, and mixtures of at leastone multi-headed amphiphile and at least one single headed amphiphile,which form vesicles with homogeneous or heterogeneous membranestructure. In any case, according to the invention, at least one of themulti-headed amphiphiles is a bolaamphiphile.

In particular embodiments, the nano-sized particles comprise a mix ofbolaamphiphiles with different headgroups. In more particularembodiments, these bolaamphiphiles comprise different alkyl cholinederivatives.

In a specific embodiment, the nanoparticle of the invention comprise amixture of Derivative 1 and Derivative 4, preferably in the ratio 2:1,respectively. These bolaamphiphilic derivatives differ in the way theacetylcholine head group is linked to the aliphatic chain. Nanoparticlescomprising said mixture loaded with a biologically active agent such asenkephalin released the encapsulated material more slowly compared tonanoparticle based only on Derivative 4, due to the mush less efficientcleavage of the acetylcholine group of Derivative 1.

In certain additional embodiments, the nanoparticles comprisebolaamphiphiles with alkyl choline headgroups and bolaamphiphiles withamino acid headgroups. Mixtures of different bolaamphiphiles usuallyform vesicles with heterogeneous or homogeneous monolayer membrane.

A mixture of bolaamphiphiles and single headed amphiphiles may formvesicles with a mosaic membrane morphology, most often a monolayermembrane formed by bolaamphiphiles, containing bilayer domainsdistributed therein formed by the single-headed amphiphiles. A mixtureof bolaamphiphiles and triple-headed amphiphiles may give raise toheterogeneous monolayer membranes as well. The relationship between thechemical composition of the different amphiphiles and theircompatibility to form homogeneous membranes vs mosaics is based on wellestablished molecular relationships and may be used by one skilled inthe art.

Preferably, the active agents comprise functional groups that mayinteract with amphiphilic headgroups and/or with other groups on theamphiphiles such as hydroxyl or other hydrogen bonding groups,hydrophobic moieties and the like.

Bioactive agents which may be delivered by the nano-particles of thepresent invention include, without being limited to, natural orsynthetic peptides, proteins, nucleosides, and polynucleotides,antiviral and antibacterial agents, e.g., antibiotics, antineoplasticand chemotherapeutic agents, and anti-inflammatory drugs. Non-limitingexamples of peptides and proteins include analgesics peptides from theenkephalin class, calcitonin, cyclosporin, insulin, insulin analogs,oxytocin, tyrotropin releasing hormone, follicle stimulating hormone,luteinizing hormone, vasopressin and vasopressin analogs, catalase,superoxide dismutase, interleukin-II, interferon, colony stimulatingfactor, tumor necrosis factor (TNF), and melanocyte-stimulating hormone.

In certain embodiments, the peptides or proteins are selected from theglial cell line derived neurotrophic factor (GDNF) or the Gly-Leu-Phe(GLF) families.

GDNF is a highly conserved neurotrophic factor that is a distant memberof the TGF beta superfamily. The GDNF gene product is processed to adisulphide-linked homodimer, which is a ligand for the RETprotooncogene. GDNF protects and repairs dopamine-containing neurons,which degenerate in Parkinson's disease, and motor neurons, which die inamyotrophic lateral sclerosis. The use of GDNF in the treatment ofParkinson's disease has shown promise in the clinic. Treatment of spinalcord injuries with GDNF has also produced neurological improvement.

The GLF peptide is an immunostimulating peptide derived fromα-lactalbumin, and was shown to prevent alopecia induced by anticanceragents.

In particular embodiments, polynucleotides selected from DNA or RNAfragments are delivered by the nanoparticles of the invention. In a moreparticular embodiment, the polynucleotide is a small interfering RNA(siRNA), a double-stranded RNA molecule of 20-25 nucleotides. siRNAsplay a variety of roles in biology. Most notably, siRNAs are involved inthe RNA interference (RNAi) pathway, where they interfere with theexpression of a specific gene. In addition to their role in the RNAipathway, siRNAs also act in RNAi-related pathways, e.g., as an antiviralmechanism or in shaping the chromatin structure of a genome. Some nonlimiting examples for target genes, or biological pathways which can beinterfered by siRNA are epidermal growth factor receptor variant IIIgene, which is expressed in 40-50% of gliomas, and the phosphoinositide3-kinase (PI3K)/Akt pathway, which plays a crucial role inmedulloblastoma biology.

In additional particular embodiment, the DNA fragment delivered by thenanoparticle of the invention is a DNA plasmid such as the BGFP-N1reporter gene.

In particular embodiments, the nano-sized particles of the invention aredesigned for delivering agents for the treatment or diagnosis ofdiseases or disorders associated with the CNS, particularlyneurological/neurodegenerative diseases or disorders such as Parkinson'sdisease or Alzheimer's disease, or for treatment of brain tumors.According to these embodiments, the nanoparticles comprise multi-headedamphiphiles, preferably bolaamphiphiles, containing headgroups that arehydrolyzed or rearranged by degradative enzymes such as hydrolases,esterases, oxidases, decarboxylases, deaminases and isomerases. Thedegradative enzymes are either restricted to the brain, oralternatively, the delivery is applied in combination with enzymeinhibitors that do not penetrate the blood-brain-barrier, therebypreventing the premature disruption of the vesicles in the peripherywhere the degradative enzyme is inhibited. For example, thenanoparticles may contain headgroups which are derivatives of choline orthiocholine, or an aromatic amino acid-type compound and the peripheralenzyme inhibitors may be a choline esterase inhibitor, an aromaticL-amino acid decarboxylase inhibitor, a monoamine oxidase (MAO)inhibitor or a catechol-o-methyltransferase (COMT) inhibitor.

For the treatment of Parkinson's disease, the drugs to be deliveredinclude, but are not limited to, levodopa (L-DOPA), carbidopa/levodopa,apomorphine, dopamine, and growth factors such as GDNF. By way ofexample, for delivery of levodopa, which is a negatively chargedmolecule due to a carboxylic group, the nanoparticle delivery vehiclewill comprise amphiphiles containing cationic headgroups at a pH abovethe pKa of the levodopa that will form vesicles complexes with enhancedencapsulation resulting partially from encapsulation within the vesiclescores as well as salt complexes formation due to ionic interactionsbetween the drug and the oppositely charged headgroups on the outersurface of the vesicles.

For the treatment of Alzheimer's disease, the drugs to be deliveredinclude, but are not limited to, antibodies against components of theAlzheimer plaques, anti-inflammatory agents, growth factors, andmuscarinic agonists that do not penetrate the BBB such as carbachol.

In particular embodiments, the nano-sized particles of the invention aredesigned for delivering agents for the treatment of cancer.Antineoplastic and chemotherapeutic agents that can be used include,without limitation, doxorubicin, cyclosporin, epirubicin, bleomycin,cisplatin, carboplatin, vinca alkaloids, e.g. vincristine,Podophyllotoxin, taxanes, e.g. Taxol and Docetaxel, and topoisomeraseinhibitors, e.g. irinotecan, topotecan.

According to these embodiments, the therapeutic agent is primarilyencapsulated in stable vesicles by known methods, e.g. active loading.In addition, substantial amounts of non-encapsulated and ionicallycharged drug may further be associated with oppositely charged groups onthe outer surface of the vesicular membrane. In more particularembodiments, the vesicles are formed from bolaamphiphiles comprisingaromatic L-amino acids headgroups which are presented on the outervesicular surface (e.g. phenylalanine, tyrosine, levodopa, tryptophan orderivatives thereof). The carboxyl group of these aromatic amino acidsis selectively cleaved by aromatic L-amino acid decarboxylase (AADC) inthe brain, thus releasing the therapeutic drug in the brain. Accordingto these embodiments, the nanoparticles are administered to a patient inneed thereof in combination with a peripheral aromatic L-amino aciddecarboxylase inhibitor (e.g. benserazide or carbidopa).

In certain additional embodiments, the surface ionic headgroups of thevesicular membrane are composed of one or more (thio)choline esters suchas acetyl, butanoyl and hexanoyl choline/thiocholine, and the peripheralactivity of cholinesterases (e.g., AChE present in the serum, liver andpancreas) is inhibited by quaternary cholinesterase inhibitors that donot penetrate the blood-brain barrier, such as neostigmine andpyridostigmine. Pyridostigmine is a carbamate used in humans for thetreatment of myasthenia gravis due to its ability to inhibitacetylcholine esterase (AChE) without penetrating into the brain.

In certain additional embodiments, the surface ionic headgroups of thenanoparticles are dicarboxylic amino acids such as glutamic acid andaspartic acid that enhance transport through the BBB and decarboxylateby various enzymes in the CNS.

The hydrolyzing or degrading enzymes inhibitors mentioned above areusually administered to the patient about 15 min up to about 1 hour or 2hours before the nanoparticles containing the drug are administered.

In certain embodiments, the nano-sized particles of the invention aredesigned for delivering active agents that exert their action in theblood circulation, have a short lifetime in the intestine and stomachand are poorly absorbed in the gastro-intestinal (GI) tract. Accordingto these embodiments, the nanoparticles are designed to contain polarheadgroups on the surface of the vesicular membrane, preferably cholineesters that are hydrolyzed by cholinesterases in the blood, thusreleasing the therapeutic agent in the blood circulation.

In particular embodiments, such nanoparticles are used for the treatmentof diabetes, and may contain insulin. In other particular embodiments,these nano-sized particles are used for the treatment of multiplesclerosis and may contain, for example, Cop 1 (Copaxone), or be used forthe treatment of breast cancer and contain antibodies such as Herceptin.Nano-sized particles for the treatment of immunodeficiency diseases maycontain a mixture of immunoglobulins. No enzyme inhibitors are needed inthese cases.

In certain embodiments, the invention provides nano-sized particlesdesigned to deliver an antibacterial or antiviral agent for theselective treatment of viral and bacterial infections. According tothese embodiments, the outer surface of the nanoparticles may containfunctional groups (as headgroups or pendants on the outer vesicularmembrane) that specifically interact with the viral wall such as, butnot limited to, lectins and inactines, or with the bacterial wall suchas, but not limited to, antibodies against the sequence LPXTG, whichconstitutes the cell wall sorting signals in a variety of bacteria aswell as specific antibodies such as those against protein A ofStaphylococcus aureus.

In certain embodiments, the invention provides nano-sized particles forthe delivery of nucleic acids/genes for gene therapy. For example,liposome formulations comprising liposome-DNA combinations/complexes forintratracheal gene therapy of lung cancer, ovarian and other cancers aswell as for gene therapy of hemophilia, and other diseases.

For the purpose of administering drugs to the brain, the nanoparticlesof the invention may contain, besides the aforementioned selectivelycleavable headgroups, ligands of specific receptors at the target site,presented as functional groups or pendants on the surface of thenanoparticles for targeting purposes, and/or ligands and surface groupswhich increase permeability through the BBB.

Examples of ligands for targeting purposes include nicotine, cytisine(nicotinic agonist), lobeline (nicotinic agonist), 1-glutamic acid (aligand of the NMDA and AMPA receptors, since it has specific transporterthat transfers it across the BBB), MK801 (NMDA antagonist), morphine(binds to the opiate receptors), enkephalins (pentapeptides that bindopiate receptors, can also be used as a headgroup that is cleavedspecifically by a brain-specific peptidase called enkephalinase),benzodiazepines such as diazepam (valium) and librium (bind to the GABAreceptor complex), dopamine agonists (e.g. bromokriptine, pergolide,ropirinol and the like), dopamine antagonists (neuroleptics such ashalidol, benzamine (sulpiride), phenothiazines), tricyclicantidepressants (such as amytyptiline and desimipramine), muscarinicagonists (such as oxotremorine, pilocarpine andcis-2-methylspiro[1,3-oxathiolane-5,3′-quinuclidine], muscarinicantagonists (have very high affinity to the muscarinic receptors, suchas atropine and scopolamine), cannabinoids such as delta-9-tetrahydrocanabbinol (delta-9-THC) and arachidonyl ethanol amide.

Other additives which may be added to enhance BBB penetration arepolycationic polymers such as polyethylene amine. Additional cationicsurface groups to be introduced will include moieties based onprotamine, polylysine or polyarginine, which have been shown to increaseBBB penetrability, and peptides and proteins which are known to enhancetransport through the BBB such as OX 26, transferrins, polybrene,histone, cationic dendrimer, synthetic peptides and polymyxin Bnonapeptide (PMBN).

Other additives used according to the invention for improving brain drugdelivery, include modified proteins or antibodies that undergoabsorptive-mediated or receptor-mediated transcytosis through theblood-brain barrier, such as bradykinin B2 agonist RMP-7 or monoclonalantibody to the transferrin receptor. Other ligands are monosaccharidessuch as glucose, mannose, ascorbic acid and derivatives thereof, forexample, glucose derivatives which use the glucose transporters GLUT-1and p-aminophenyl-alpha-mannopyranoside.

Additives which enhance transport across membranes of the intestinaltract are also encompassed by the present invention. Such additivesinclude, but are not limited to, chitosan (CS) and derivatives ofchitosan, Ca²⁺ chelators, medium-chain fatty acids or glycerides,steroidal detergents, and other mucoadhesive polymers.

The concentration of the various additives is readily adjusted accordingto experimental determination.

Chitosan (CS) and derivatives thereof are known intestinal absorptionenhancers which are able to increase the paracellular permeability ofpeptide drugs across mucosal epithelia. The present inventors have shownthat CS and particularly a novel CS derivative vernolyl-chitosan, aconjugate of CS and vernolic acid, is a very efficient penetrant of theBBB as well as of membranes of the GI tract and mucosal membranes.

Chitosan is a linear polysaccharide composed of randomly distributedβ-(1-4)-linked D-glucosamine (deacetylated unit) andN-acetyl-D-glucosamine (acetylated unit). It is produced commercially bydeacetylation of chitin, which is the structural element in theexoskeleton of crustaceans (crabs, shrimp, and the lime) and cell wallsof fungi. The properties of CS and CS derivatives that make themimportant additives are their ability of adhering to the mucosal surfaceand transiently opening the tight junctions between epithelial cells(Silva et al., 1994) (“Microencapsulation of hemoglobin inchitosan-coated alginate microspheres prepared byemulsification/internal gelation”, Silva et al. António J. Ribeiro, 2Margarida Figueiredo, 3 Domingos Ferreira, 4 and Francisco VeigaPharm.Res. 1994; 11:1358-1361).

Other chitosan and chitosan derivatives used in accordance with thepresent invention are commercially available CS or CS reduced inmolecular weight (MW) by various processes (e.g., depolymerization of acommercially available CS using enzymatic degradation with cellulose,followed by precipitation at ˜pH 7.0 and derivatization e.g., byquaternization of the amino groups with methyl iodide, conjugatingchitosan with propylene or ethylene glycol to obtainchitosan-polypropylene glycol (PPG) and chitosan-polyethylene glycol(PEG) conjugates, respectively, and other derivatives known in the art.

In a particular embodiment, a novel chitosan-PEG additive, CS-PEG₂₀₀₀ isused.

The aforementioned derivatives of chitosan are generally made byreaction with either the hydroxyl and/or amino groups of the chitosanpolymer. The two hydroxyl groups have slightly different reactivity butcan be functionalized by hydroxy active agents at high pH on either theacetylated or deacetylated monomers of the chitosan. The primary amineof the deacetylated monomer is available for reaction at moderate pHabove 6 or so where a significant number of the amines are deprotonated.These chemistries provide new chitosan compounds bearing differentproperties from the original chitosan polymer.

Examples of chitosan derivatives made by reaction of the hydroxyl groupsinclude, but are not limited to, carboxyalkylated chitosan, sulfonylchitosan, carbohydrate-branched N-(carboxymethylidene) chitosan andN-(carboxymethyl) chitosan.

Many derivatives of chitosan are related to reactions of the aminogroups on the glucosamine units, for example quaternary ammoniumderivatives. Methods for synthesis of quaternary ammonium derivativesare well known in the art. Another example is derivatives resulting fromcoupling of the amino groups to carboxylic acids using peptide couplingchemistry. Known derivatives of low molecular weight chitosan polymers(less than 10,000 Da) N-conjugated with different amino acids include,but not limited to, chitosan-asparagine, -glycine, -alanine, -asparticacid, -cysteine, -methionine and chitosan-arginine, wherein the aminoacid is bound through a peptidic (amide) bond via its carbonyl to theprimary amine on the glucosamines of chitosan.

Low molecular weight chitosan derivatives of about 80 kDa, preferablyless than 30 kDa, are preferred according to the invention for thepurpose of enhancing paracellular transport. CS of 30-50 kDa is kidneyinert.

In particular embodiments, the present invention provides nanoparticlescomprising at least one bolaamphiphile selected from the hereindesignated Derivative 1, Derivative 2, Derivative 3 and Derivative 4, anactive agent selected from leu-enkephalin, carboxyfluorescein, ¹²⁵I-GDNFand ovalbumin and at least one additive selected from vernolyl chitosan,Derivative 5, PEG-vernonia conjugate, cholesterol and cholesterylhemisuccinate.

In another aspect, the present invention provides bolaamphiphiles of theformula Ia and IIa above. In certain embodiments, bolaamphiphiles of theformula IIa are provided comprising a —CO—R₁₀—CO— midsection. Inparticular embodiments, these bolaamphiphiles have a hydrogen-bondinggroup —OH located adjacent to the cleavable headgroup on the vicinalcarbon atom (positions 1-2), which contributes to the stabilization ofthe nanoparticles based thereon. In more particular embodiments, thesebolaamphiphiles are symmetric bolaamphiphiles selected from thecompounds herein designated Derivative 1 and Derivative 5, or theasymmetric bolaamphiphile herein designated Derivative 3.

In other particular embodiments, symmetric or asymmetric bolaamphiphilesof the formula Ia are provided, which comprise the midsection—N(H)—R₁₀—N(H)— and a hydrogen bonding moiety located adjacent to thecleavable headgroup or the headgroup containing the cleavable group ormoiety. In a more particular embodiment, the bolaamphiphile is theasymmetric compound herein designated Derivative 2, which comprises aterminal acetylcholine head group and a non-terminal acetylcholineheadgroup located adjacent to an OH group (positions 1-2).

In certain embodiment, the present invention provides bolaamphiphiles,which do not have a hydrogen-bonding group located adjacent to thecleavable headgroup. These bolaamphiphiles may be prepared fromricinoleic acid based on castor oil, lesquerella oil and jojoba oil.

In particular embodiments, the bolaamphiphiles are symmetricbolaamphiphiles of the formula IIa. In more particular embodiments,these bolaamphiphiles are selected from the compounds herein designatedDerivative 6, Derivative 7 and Derivative 8.

In further particular embodiments, the bolaamphiphiles are asymmetricbolaamphiphiles of the formula IIa. In more particular embodiments,these bolaamphiphiles are selected from the compounds herein designatedDerivative 9 and Derivative 10.

In another aspect, the present invention provides pharmaceuticalcompositions comprising nano-sized particles of the invention and apharmaceutically acceptable carrier. The pharmaceutical composition ofthe invention can be delivered by any suitable route including, but notbeing limited to, intravenous, intramuscular, subcutaneous, orintraperitoneal injections, oral, nasal, lung or gum administration.

In certain embodiments, the pharmaceutical compositions are formulationsfor oral uptake. In certain additional embodiments, the formulations areinjectable formulations for i.v. administration.

The oral formulations of the present invention preferably compriseagents that enhance penetration through the membranes of the GI tractand enable passage of intact nanoparticles containing the drug. Theseagents may be any of the additives mentioned above, preferably chitosanand derivatives thereof, serving as vehicle surface ligands, preferablyas decorations or pendants on the vesicles, or the agents may beexcipients added to the formulation.

In a further aspect, the present invention relates to the use of anano-sized particle as described above for treatment or diagnosis ofdiseases or disorders selected from: (i) a disease or disorderassociated with the CNS, particularly neurological/neurodegenerativediseases or disorders such as Parkinson's disease, Alzheimer's diseaseor multiple sclerosis; (ii) cancer such as breast cancer, prostatecancer and brain tumors; (iii) diabetes; (iv) an immunodeficiencydisease; and (v) viral and bacterial infections.

In particular embodiments, the nano-sized particles of the invention areused for gene therapy.

In still another aspect, the present invention provides a method fortreatment of a disease or disorder selected from: (i) a disease ordisorder associated with the CNS, particularlyneurological/neurodegenerative diseases or disorders such as Parkinson'sdisease, Alzheimer's disease or multiple sclerosis; (ii) cancer such asbreast cancer and brain tumors; (iii) diabetes; (iv) an immunodeficiencydisease; and (v) viral and bacterial infections, comprisingadministering to an individual in need thereof a nano-sized particle ofthe invention. When the target site of the drug encapsulated within thenanoparticle is the CNS, the nanoparticle is preferably administeredtogether with a suitable peripheral enzyme inhibitor thus preventingpremature disruption of the nanoparticle and release of the active agentoutside the desired biological target site.

In a particular embodiment, the present invention provides a method forgene therapy.

EXAMPLES Chemical Section Materials

Vernonia oil, containing an average of 2.1 epoxy groups per molecule ofoil, was purchased from Ver-Tech, Inc., Bethesda, Md.5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB); Acetylcholinesterase(AChE) EC3.1.1.7, type V-S lyophilized from Electrophorus electricus(electric eel), catalogue No.: C-2888; Acetylthiocholine (ATC) iodide;5(6)-Carboxyfluorescein (CF); Pyridostigmine(3-(Dimethylamino-carbonyloxy)-1-methylpyridinium bromide); Triton®X-100 (t-Octylphenoxy-polyethoxyethanol); Triton® X-100—Reduced form;Uranyl acetate dehydrate; Cholesterol (5-Cholesten-3β-ol); [5-Leucine]Enkephalin; L-α-Phosphatidylcholine Distearoyl (C18:0) (DSPC);Cholesteryl Hemisuccinate (5-Cholesten-3β-ol 3-hemisuccinate); SephadexG-50; Trizma® Base (Tris {hydroxymethyl}aminomethane) and itshydrochloride salt; Perchloric acid (PCA), Trichloro acetic acid (TCA)and 2,2,2-Tribromoethanol (Avertin), all were of analytical grade andpurchased from Sigma-Aldrich® Inc.1,2-Dioleoyl-3Trimethylammonium-Propane(Methyl Sulfate Salt) (DOTAP) waspurchased from Avanti Polar Lipids, Inc. Morphine HCl 20 mg/ml waspurchased from Teva Pharmaceuticals Industries Ltd. Na₂HPO₄ and KH₂PO₄were purchased from Biolab Ltd. ³H-cholesteryl oleyl ether ([1 alpha, 2alpha (n)-³H]Cholesteryl oleyl ether, 30-60 Ci/mmol, 1 mCi/ml), waspurchased from Amersham Biosciences Inc. (UK). Solutions for inducinganastasia in animals—Ketamine HCl 100 mg/ml and Xylazine 2%, wereobtained from the BGU's animal facility. Other standard chemicals wereall purchased from commercial sources.

Methods

i. Vesicle Formation

The methods used for nanoparticle preparation are well known in the art(see for example, New R.R.C. (1990) Preparation of liposomes In:Liposomes: A Practical Approach. IRL Press, Oxford) and include ethanolinjection optionally followed by sonication or extrusion, andfilm-hydration-sonication where the sonication may be probe sonicationor bath sonication.

Nanoparticle formation was conducted at room temperature (about 25° C.),which is above the transition point of the bolaamphiphilic compoundsused in the present study. The preparation of liposomes from DSPC wasdone by FHE at 65° C., which is above the transition point of DSPC.

a. Vesicle Formation by Ethanol Injection (EI)

The following method was typically used for obtaining vesiclesuspensions without additives:

Principle: the bolaamphiphile is dissolved in ethanol and the solutionthen rapidly injected through a fine needle below the surface of astirring aqueous solution containing the water-soluble material to beencapsulated (i.e. CF or leu-enkephalin). The force of the injection andthe stirring are sufficient to achieve complete and rapid mixing, sothat the ethanol is diluted almost instantly in the aqueous phase andthe lipid molecules are dispersed evenly throughout the medium. Toassure good mixing and vesicle formation, a sonication step is performedfollowing the injection of the bolaamphiphile ethanolic solution.

Standard procedure: 10 mg of a bolaamphiphile was dissolved in 50 μlethanol. The following steps were done in the dark: 1 ml of the materialto be encapsulated (dissolved in the relevant medium) was added to a 5ml glass vial and stirred vigorously while the bolaamphiphile sample wasinjected thereto as quickly as possible through a HPLC syringe. Themixture was let to stir for 1-2 minutes, and then sonicated in Elma bathsonicator (50/60 Hz), 30 minutes at RT.

b. Vesicle Formation by Film-Hydration-Sonication (FHS) andFilm-Hydration-Extrusion (FHE)

The following method was typically used for obtaining nanoparticles withadditives. Unless mentioned otherwise, the molar ratio ofbolaamphiphile:cholesteryl hemisuccinate:cholesterol was 2:1:1,respectively. When PEG-vernonia conjugates, was used as additive, theratio of the bolaamphiphile to the PEG-vernolic acid conjugate was 10:1,respectively.

The bolaamphiphiles (10 mg) and the additives (in respective amounts)were dissolved in an organic solvent (chloroform, 750μ) using vortex.The solution was then placed in a 50 ml round bottom flask and heldunder vacuum, while rotating, at a rate of 90/min in a Rotarvap for1-1.5 hours until all the solvent was completely evaporated. A driedthin film of the bolaamphiphile lining the walls of the flask was formedwhen the evaporation step was completed. Then, the thin film washydrated by adding 1 ml of a solution containing the material to beencapsulated and mixed until all the film was dissolved. The solutionwas then either bath sonicated for 30 minutes at RT (FHS), or extruded(FHE) via 0.1/0.2 μm polycarbonate membrane (GE Water & ProcessTechnologies, purchased from Tamar Laboratory Supplies Ltd.) till thesolution became transparent (approx. 8-10 times).

When CS pendant were incorporated into the nanoparticles, probesonication was used rather than bath sonication and the method of thevesicle preparation was modified as described below. Unless mentionedotherwise, the molar ratio for CS was 10% relative to thebolaamphiphile.

Ten (10) mg of the bolaamphiphiles and other additives (in respectiveamounts) except for the CS-vernolic acid conjugate were dissolved inchloroform, evaporated to form a thin film and rehydrated as describedabove. Then 1 ml of the material to be encapsulated dissolved in therelevant medium and containing 1 mg CS-vernolic acid conjugate was addedand mixed until all the film was dissolved. The solution was placed in a5 ml glass vial and sonicated in a probe sonicator (Vibra Cell ModelH540/CV54, Sonics and Materials U.S.A) for 15 minutes in ice-coldcontainer under the following conditions: 21% amplitude; pulse mode incycles: 15 seconds pulse/10 seconds rest.

Under these conditions, the CS-vernolic acid conjugate is incorporatedinto the membrane of the vesicles and the CS moiety remains as pendanton the vesicle surface.

Example 1 Synthesis of Vernoyl Chitosan

The use of chitosan is limited due to its insolubility at pH higher than6. One of the most common ways of providing hydrophilic characteristicsto this polysaccharide is depolymerization.

i. Degradation of Chitosan by Hydrogen Peroxide

The oxidative degradation of chitosan was performed with hydrogenperoxide according to methods known in the art (e.g., Wu et al., 2005),using chitosan of an average molecular weight of 5-10 kDa, as follows:

Chitosan (5 g, Mn=54 KDa, degree of deacetylation 70-80%) was dispersedin 150 ml of water at RT for 1 hour. A solution of hydrogen peroxide (5ml, 30%) was added dropwise to the chitosan dispersion and the reactionmixture was heated at 60° C. for 6 hours. The pH of the solution wasthen adjusted to pH=7 with a 1 M NaOH solution. The insoluble chitosanwas filtered, the filtrate was evaporated, and absolute ethanol wasadded to the residue to precipitate the product that was repeatedlywashed with absolute ethanol, and dried under vacuum to give 2.5 g (50%yield) of the water soluble chitosan oligomer.

The weight-average molecular weight (Mw), number-average molecularweight (Mn), and molecular weight dispersion Mw/Mn were measured by GPCin 0.2 mol/l CH₃COOH/0.1 mol/l CH₃COONa solutions as the eluent, using aTSKgel G4000PWXD column with a refractive index detector. The watersoluble chitosan had Mn˜1500-2000 Da, Mw=2800-3500 Da, andMw/Mn=1.7-2.1.

Elemental analysis (%): C, 38.88; H, 6.63; N, 6.12. The amount of NH₂groups was about 45-50% and the amount of carboxylic groups determinedby titration with NaOH was 1.1 mmol/g.

According to MALDI-TOF mass spectrometry the soluble chitosan obtainedhad the following structure:

The water soluble product was also characterized by FT-IR spectroscopy.FT-IR spectra showed that the intensity of the absorption band at 1593cm⁻¹ characteristic of NH₂ in the degraded chitosan has higher intensitycompared to the starting material.

ii. Preparation of Vernolic Acid N-Hydroxysuccinimide (Ver-NHS)

Ver-NHS was prepared following the procedure of Lapidot et al. (1967):to a mixture containing stoichiometric amounts of vernolic acid (2.4597g, 0.00831 mol) and N-hydroxysuccinimide (0.9564 g, 0.00831 mol) in 40ml of dry ethyl acetate, N,N′-dicyclohexylcarbodiimide (DCC; 1.7146 g,0.00831 mol) in 5 ml of dry ethyl acetate was added. The mixture wasstirred overnight at RT, the dicyclohexylurea (DCU) formed was filtered,and the solvent was removed under reduced pressure to yield the crudematerial comprising Ver-NHS and residual DCC and DCU. The pure Ver-NHS(1.5 g, Rf=0.2) was separated by flash-chromatography with a mixture ofpetroleum ether (60-80):diethyl ether 1:1 (v/v) as the eluent. Yield:48%

iii. Preparation of Vernoyl Chitosan

The water-soluble chitosan obtained in (i) above, was reacted withVer-NHS in order to covalently bind the vernonia moiety to the chitosan,as follows:

Chitosan obtained after treatment with hydrogen peroxide (0.2543 g,0.8718 mmol of the amino groups of chitosan) was dissolved at 60-70° C.in dry DMSO. The solution was cooled to RT and triethyl amine was added.A 5.0 ml solution of Ver-NHS (0.0686 g, 0.1745 mmol) in dry DMSO wasdropwise added to the chitosan solution and the reaction mixture wasstirred at RT for 72 h. The triethyl amine was removed by evaporationand the remaining clear solution was freeze-dried. The dried powder waswashed with diethyl ether, several times with ethanol and dried in avacuum desicator at RT. A light-yellow powder was obtained. The productwas characterized by FT-IR and ¹³C-NMR (d₆-DMSO).

Example 2 Synthesis of Vernolyl Glycol Chitosan

The water-solubility of chitosan (CS) can be substantially enhanced byattaching glycol units to it. Vernolyl glycol chitosan serves as anadditive that enhances the penetrability and stability of thenanoparticles of the invention.

Vernolyl glycol chitosan was prepared based on the process of Kwon etal. (2003) for the preparation of hydrophobically modified glycolchitosans (HGCs), by the covalent attachment of vernolic acidN-hydroxysuccinimide (Ver-NHS) to glycol chitosan via an amide bond to afree amino group of a glucosamine unit. The synthesis of vernolyl glycolchitosan starting from glycol chitosan is depicted in Scheme 1.

A solution of Ver-NHS (0.1133 g, 0.000283 mol), prepared according toExample 1, in 60 ml of absolute ethanol was dropwise added to 40 ml of a0.055 N NaHCO₃ solution of glycol chitosan (0.2364 g, 0.001153 mol). Themixture was stirred for 72 h in darkness. The reaction mixture wasconcentrated by reducing the solvent under reduced pressure at 45° C.down to a volume of 40 ml. The resulting aqueous dispersion of thepolymer was extracted with diethyl ether (3×100 ml) and left to standfor 24 h in a fume hood for evaporating residual ether, then dialyzedagainst water (5 l) over 24 h, and finally freeze-dried to give acotton-like-solid.

The polymer obtained was of ˜400 units, MW of a unit 205 Da.

Different ratios of chitosan and Ver-NHS (5:1; 8:1; 10:1) were used tostudy the degree of substitution of the product and its properties aspenetration enhancers via the intestinal membrane. The study ofdifferent rations of the components that make up the nanoparticle isneeded to achieve an optimum composition of components for stability,penetrability through different biological barriers and a selectivedisruption at the target site.

Example 3 Preparation of PEG-Vernonia Conjugates

PEG derivatives of vernolic acid are used as additives in thepreparation of the nanoparticles of the invention and were synthesizedas follows:

i. Synthesis of PEG₂₀₀₀-Vernonia Conjugate

Two kinds of PEG-vernonia derivatives were prepared. The first kind wasobtained by opening the epoxy ring and binding PEG to the oxygen atomvia an ether bond (see Scheme 2). These derivatives are termed herein“PEG-ether derivatives” or “PEG(202)”.

The second kind was obtained through an enzymatic reaction betweenvernolic acid and PEG to form an ester bond at the carboxylic group ofvernolic acid, as shown in Scheme 3. These derivatives are termed herein“PEG-ester derivatives” or PEG(201)”.

PEG₂₀₀₀-Ether Derivative (PEG(202))

To a mixture containing 12.95 gr (0.006452 mol) of PEG₂₀₀₀ and BF₃etherate (600 μl), 2 gr (0.006452 mol) of methyl vernolate was injectedevery 5 to 10 minutes to in portions of 0.2 ml. The reaction mixture wasrefluxed at 80-86° C. for 7 hours. After cooling to RT, the reactionmixture was dissolved in ether and filtered. The crude product waspurified by flash column chromatography using a mixture ofchloroform:methanol as the eluent.

PEG₂₀₀₀-Ester Derivative (PEG(201))

A mixture containing 1 gr (0.0034 mol) vernolic acid, 10 gr PEG₂₀₀₀(0.005 mol) and 0.0688 gr Candida antarctica (Novozym 435) lipase intoluene was refluxed at 75-80° C. for 7 hours. The reaction mixture wascooled, the lipase was and filtered and the solvent was removed underreduced pressure. The crude product was purified by flash columnchromatography using a mixture of chloroform:methanol as the eluent.Both kind of derivatives were characterized by FT-IR, NMR and MALDI

Example 4 Synthesis of Derivative 1

The synthesis of Derivative 1 is schematically shown in Scheme 4.

i. Synthesis of Precursor 1 (Decane Divernolate)

A mixture of 92.1 g (0.31 mol) of vernolic acid and 27.5 g (0.155 mol)of 1,10-decandiol was refluxed with 0.77 g of lipase acrylic resin fromCandida antarctica (Novozym 435) in 300 ml of toluene. Water formedduring the reaction was removed by azeotropic distillation under reducedpressure (130-170 mm Hg). The temperature in the oil bath was held at75-90° C. After 5 h, the reaction mixture was cooled, the lipase wasfiltered off, and the reaction mixture was concentrated to about 20 mlThereafter, 400 ml of methanol were added, and the suspension obtainedwas left to stand overnight in a refrigerator. The precipitate wasfiltered off and washed with cold methanol to give 96.2 g of Precursor 1in 85.7% yield (purity 89% HPLC).

(ii) Synthesis of Precursor 2 (Decane Divernolate Diglutaric Acid)

Precursor 2 was obtained by opening of the epoxy rings of the vernolatemoieties of Precursor 1 with glutaric acid, as follows.

A mixture of 6.1 g (8.3 mmol) of decane divernolate and 16.5 g ofglutaric acid in 50 ml of 1,2-dichloroethane were refluxed for 48 h.After cooling the reaction mixture, 200 ml of chloroform were added, andthe solution was washed with a saturated solution of NaCl until the pHreached 6. The chloroform solution was dried over magnesium sulfate andfiltered, and the solvent was removed under reduced pressure to give 7.8g of crude Precursor 2. Purification of the crude product by columnchromatography on silica gel 60 using hexane:ether:acetic acid 5:5:0.1as the eluent yielded 3.5 g of pure product (41.6% yield) with 98%purity.

iii. Synthesis of Precursor 3

Precursor 3 was obtained by esterification of Precursor 2, as follows:

A solution of 10.3 g of 1-ethyl-3-(3,3-dimethylaminopropyl)carbodiimide(EDCI) in 100 ml of dry dichloromethane was added dropwise to anice-cooled solution of 50 ml of dry dichloromethane containing 12.9 g(0.013 mol) of Precursor 2, 8.85 g of 4-dimethylaminopyridine (DMAP),and 16 ml of N,N-dimethyl amino ethanol. The reaction mixture wasstirred for two days, and chloroform was then added. The organicsolution was washed several times with a saturated NaCl solution untilthe pH reached 7. The organic solvent was dried over anhydrous magnesiumsulfate and the solvent was removed under reduced pressure to yield 9 gof crude product (70% HPLC, methanol:water (0.15% trifluorocicetic acid)95:5; Rt-6.9 min, flow 0.5 ml/min, TLC chloroform:methanol 8:2). Theproduct, Precursor 3, was purified by flash column chromatography withacetone as the eluent.

iv. Synthesis of Derivative 1.

Precursor 3 was quaternized to obtain the symmetric bolaamphiphileDerivative 1 as follows:

A mixture of 0.7 g Precursor 3 and 1 ml of CH₃I in 20 ml of drydichloromethane was stirred for 24 h in a cooling bath. The solvent wasremoved under reduced pressure, and the iodide was exchanged withchloride on an ion-exchange resin (Amberlyst CG-400-I) to yield 0.67 gof the pure product Derivative 1 (see Scheme 4).

Example 5 Synthesis of Derivative 2

The asymmetric bolaamphiphile Derivative 2 was synthesized starting fromthe synthesis of vernol monoaminoamide as follows:

i. Synthesis of Precursor 5 (Vernol Monoaminoamide)

A mixture containing 3.1 g (0.0155 mol) of 1,12-diaminododecane in 30 mlof dry toluene and 2.0 ml (0.001 mol) of a 0.5 N sodium methoxidesolution in absolute ethanol was heated to 70° C. To this solution, 4.8g (0.0155 mol) of methyl vernolate in 20 ml of toluene were dropwiseadded during 3.5 h, and the reaction mixture was heated for two morehours. After cooling, the solidified reaction mixture was trituratedwith hexane, filtered and washed with water until pH=7. The crudeproduct was purified by column chromatography on Silica gel using amixture of CHCl₃:CH₃OH:25% NH₄OH in the ratio 100:10:1. The purecompound (MP=124-126° C.) was characterized by FT-IR, NMR, an ESI-MS(m/z: 479.6[M+H]⁺).

ii. Synthesis of Precursor 6 (Mono-Chloroacetatae of VernolMonoaminoamide)

Into a three-neck round-bottom flask, Precursor 5 prepared in (i) abovewas introduced (0.01 mol), along with 20 ml of dry chloroform and 1.4 ml(0.01 mol) triethyl amine. The reaction mixture was cooled to −12° C.and a solution of chloroacetyl chloride (0.66 ml, 0.0083 mol) in 5 ml ofdry chloroform was dropwise added during 40 minutes. The temperature ofthe reaction raised to 10° C. After an additional hour at RT, chloroformwas added to the reaction mixture and the organic phase was washed witha 5% solution of NaHCO₃ until pH=7, and then with water until no moreCl⁻ ions could be detected. The organic phase was separated and driedover anhydrous magnesium sulfate. The solvent was removed under reducedpressure and the crude product was purified by column chromatography onsilica gel with a mixture of chloroform:acetone (8:2) as the eluent. Thepure product was obtained in a 58% yield as a white solid (MP=115-116),and was characterized by FT-IR, NMR, and ESI-MS.

iii. Synthesis of Precursor 7 (Di-Chloroacetatae of VernolMonoaminoamide)

A solution containing Precursor 6 (0.001 mol) in 4 ml of dry toluene andchloroacetic acid (0.13 g, 0.0014 mol) was heated at 75° C. for 12 h.Chloroform was added and the organic phase was washed with a 5% solutionof NaHCO₃ until pH=7, and then with water. The organic phase wasseparated and dried over anhydrous magnesium sulfate. The solvent wasremoved under reduced pressure and the crude product was purified bycolumn chromatography on silica gel with a mixture of chloroform:acetone(7:3) as the eluent. The pure product was obtained in a 44% yield as asticky white solid (MP=115-116), and was characterized by FT-IR, NMR,ESI-MS (m/z: 671.9 [M+H]⁺).

iv. Synthesis of Derivative 2

Derivative 2 was obtained by quaternization of Precursor 7, as follows:

A mixture containing Precursor 7 (0.001 mol) and 0.294 g (0.0022 mol) ofN,N-dimethylamino ethyl acetate in 2 ml of dry acetone was refluxed forabout 10 hours. after cooling to RT, 5 ml of dichloromethane and 15 mlof diethyl ether were added and the reaction mixture was refrigeratedovernight. The supernatant was separated by decantation to leave aviscous liquid. This procedure was repeated several times to remove theunreacted N,N-dimethylamino ethyl acetate. Solvent residues were removedunder reduced pressure to give the product Derivative 2 in a 48% yieldas a colorless viscous liquid that was characterized by FT-IR, NMR,ESI-MS (m/z: 420.1 [M-2Cl]²⁺/2).

¹H NMR (d₆ DMSO) δ ppm 9.15 (1H, m, NH—C(O)—CH₂—N⁺), 7.86 (1H, m,CH₂—CH₂—NH—C═O), 5.41, 5.32, (2H, 2m, CH═CH), 5.23, 5.08 (1H, 2d, J=5.5Hz, CH—OH), 4.81 (1H, m, CH—O—C═O), 4.68 (2H, m, ⁺N—CH ₂—C═O), 4.45 (4H,m, O—CH ₂—CH₂—N⁺), 4.30 (2H, s, ⁺N—CH ₂—C═O), 3.94, 3.90 (4H, 2m,O—CH₂—CH ₂—N⁺), 3.50 (1H, m, CH—OH), 3.32 (6H, m, (CH ₃)₂—N⁺), 3.28 (6H,s, (CH ₃)₂—N⁺), 3.08 (2H, q, ³J=6.3 Hz, CH₂—CH ₂—NH—C(O)—CH₂—N⁺), 2.98(2H, q, ³J=6.3 Hz, CH₂—CH ₂—NH—C(O)—CH₂—CH₂), 2.31, 2.12 (2H, 2m, CH—CH₂—CH═CH), 2.04, 2.03 (6H, 2s, CH ₃—C═O), 2.03 (2H, m, CH₂—CH ₂—C═O),1.98 (2H, m, CH═CH—CH ₂—CH₂), 1.22-1.60 (38H, m, (CH ₂)_(n)), 0.84 (3H,m, CH₂—CH ₃); ¹³C NMR (d₆ DMSO) δ ppm 171.9 (NH—C(O)—CH₂—CH₂), 169.8 and169.7 (CH₃—C═O), 164.8 and 164.6 (CH—O—C═O), 163.0 (NH—C(O)—CH₂—N⁺),132.4, 131.0 and 125.6, 124.1 (CH═CH), 78.6, 78.4 (CH—O—C═O), 70.2 and69.7 (CH—OH), 62.5 and 62.4 (O—CH₂—CH₂—N⁺), 62.3 and 61.3 (⁺N—CH₂—C═O),57.6 (O—CH—CH₂—N⁺), 51.7 and 51.5 ((CH₃)₂—N⁺), 38.5 and 38.2(CH₂—NH—C═O), 35.3 (CH₂—C═O), 32.3, 31.5, 31.2, 31.0, 30.7, 29.6-28.6,27.8, 26.8, 26.7, 26.4, 25.3, 24.8, 24.4, 22.0 and 21.9 (CH₂—CH₃), 20.6(CH₃—C═O), 13.9 and 13.8 (CH₂—CH₃); FT-IR (neat) ν_(max) 3303 (OH), 3318(NH), 3063, 3020, 2927, 2857, 1745 (C═O), 1673, 1646 and 1554 (amidebands), 1460, 1373, 1234 (acetate band), 1055, 953, 722 cm⁻¹; ESI-MSm/z: 420.1 [M-2Cl⁻]⁺²/2; Argentometric titration calcd forC₄₆H₈₈Cl₂N₄O₉: 7.77% Cl⁻ found 8.20% Cl⁻.

Example 6 Synthesis of Derivative 3

Derivative 3 was synthesized similarly to the synthesis Derivative 2starting with vernol-monohydroxyester instead of vernol monoaminoamide,via formation of the dichloro acetate of venolmonoester and thenquaternization with N,N-dimethylamino ethyl acetate to give therespective Derivative 3.

Example 7 Synthesis of Derivative 5

The symmetric bolaamphiphile Derivative 5, which bears two glutamic acidhead groups, can be used as an additive in the formation ofnanoparticles comprising asymmetric bolaamphiphiles such as Derivative 3and or symmetric Derivative 4, and constitutes only about 10% of thetotal amphiphiles' mass. Addition of this bolaamphiphile resulted innanoparticles having improved blood circulatory. Nanoparticlescomprising Derivative 5 as the major amphiphilic component can also beused for targeted delivery of active agent.

Derivative 5 was synthesized from Precursor 1 (decane divernolate) whichin turn was obtained form vernolic acid according to the proceduredescribed in Grinberg et al., 2008. The synthesis of Derivative 5 isdepicted in Scheme 6.

A mixture of protected glutamic acid Z-Glu-OBzl (1.83 g, 4.93 mmol) andPrecursor 1 (1.5 g, 2.052 mmol) was heated to melt (100-110° C.) under anitrogen atmosphere. Tetramethyl ammonium bromide (TMABr) (0.2134 g) wasadded, and the mixture was stirred and heated for about 12 hours.

After cooling, a yellow viscous liquid was separated and dissolved indiethyl ether. The TMABr that did not dissolve in the ether solution,was filtered out. The crude (2.3744 g; ˜67.5% yield) Precursor 8 waspurified with a silica gel column using a mixture of Hexane:EtOAc, 2:1as the eluent to give 0.9196 g; 30.4% yield with a 99% purity.

Catalytic hydrogenation in the presence of Pd/C removed the twoprotecting groups of each head groups in one stage and Derivative 5 wasobtained as follows:

Into a pressure resistant vessel, a solution of Precursor 8 (0.9196 g;0.621 mmol) in 25 ml MeOH, and the catalyst were added (10% Pd/C).Hydrogen was bubbled into the reactor (H₂ pressure of 40 Lb./in² wasproduced in the vessel). The hydrogenation of the product (for purposesof removing the protective groups) was carried out for about 1 hour withcontinuous shaking.

The catalyst was filtered, the solvent was removed under reducedpressure to obtain white-opaque, very tough solid flakes Derivative 5(0.4397 g; 0.42 mmol; 68.5% yield with 99.7% HPLC purity).

Example 8 Synthesis of Derivatives 6, 7 and 8

The synthesis of symmetric and asymmetric bolaamphiphilic compounds fromcastor oil based on ricinoleic acid or methyl ricinoleate with theacetylcholine head group attached through the nitrogen atom of acetylcholine is as follows.

i. Synthesis of Precursor 9 (1,10-Decandiricinoleate)

A mixture of 2.121 g (7.12 mMol) of ricinoleic acid, 0.7 g (3.6 mMol) of1,10-decandiol and 100 mg of lipase acrylic resin from candidaAntarctica in 6.9 ml of toluene was boiled. Water formed during thereaction was removed by azeotropic distillation under diminishedpressure of 130 mm-170 mm Hg. The temperature in the bath oil was 75-90°C. After 5 hours the reaction mixture was cooled, the lipase wasfiltered and the solvent was evaporated. Silica gel Columnchromatography in Petroleum Ether/Diethyl Ether 6/1, 6/4 led to 2.647 gof pure Precursor 9 as a white powder. M.P 45-47° C.

FT-IR ν_(max) 3383 (OH), 3017 (CH═CH), 2927, 2855, 1736 (O—C═O), 1461,1246, 1175, 1081 cm⁻¹.

ii. Synthesis of Precursors 10 and 11

A mixture of the diols HO—(CH₂)₂—OH and HO—(CH₂)₄—OH (1.25 mole), methylricinoleate (2 moles) and lipase acrylic resin from Candida antartica(Novozym 435) (10% of the weight of reagents) was stirred at roomtemperature in CHCl₃ overnight. The reaction was monitoring by TLC(Petroleum Ether/Ether: 1/1). The slurry mixture obtained was dissolvedin ether, the lipase was filtered off and the outcome solution wasevaporated under vacuum. The mixture of esters was purified by columnchromatography with increasing polarity Petroleum ether/Ether: 7/1; 6/4;1/1. Precursor 10 was obtained at 34.7% yield (0.35 g), and Precursor 10was obtained at 35.6% yield (0.72 g).

iii. Synthesis of Precursors 12 and 13

Precursors 12 and 13 were synthesized from methyl ricinoleate (2.58 g,8.27 mMol), and 1,2-ethandiol or (0.321 g, 5.17 mMol) or 1,10-decandiol(0.85 g, 4.91 mMol), and 4 ml CHCl₃ as described above for Precursor 11.

Precursors 12 was obtained at 65% yield (0.35 g). FT-IR ν_(max) 3396;3007; 2927; 2855; 1737; 1455; 1388; 1255; 1081; 1040; 876 cm-1.

and Precursors 13 was obtained at 60% yield (0.64 g).

FT-IR ν_(max) 3258 (OH), 3174 (CH═CH), 3070, 2956, 2852, 1735, 1461,1260, 1177, 1051, 883, 796, 721 cm-1.

iv. Synthesis of Precursors 14-18

A mixture of Precursors 9-13 (1 mole) and chloroacetyl chloride (6moles) in 2 ml of dry diethyl ether was magnetically stirred at roomtemperature for 3 hours. The reaction was monitored by TLC. The reactionmixture was dissolved in diethyl ether, washed with a solution of 5%NaHCO₃ and distilled water till pH 5.39. The organic phase wasseparated, dried with MgSO₄, filtered and the solvent was removed underreduced pressure and crude products were obtained and characterized byFT-IR, ¹H and ¹³C NMR and MS.

Precursor 14 (dichloroacetate 1,2-ethandiricinoleate) was obtained at89% yield (0.39 g).

FT-IR ν_(max) 3017, 2954, 2928, 2856, 1756 (COCH₂Cl), 1741, 1466, 1369,1285 (CH₂Cl), 1260, 1171, 1037, 797 cm⁻¹.

Precursor 15 (dichloroacetate 1,4-butandiricinoleate) was obtained at90% yield (0.80 g).

FT-IR ν_(max) 3017, 2926, 2854, 1756 (COCH₂Cl), 1734, 1455, 1365, 1312,1286 (CH₂Cl), 1180, 1091, 1030, 876, 805 cm-¹.

Precursor 16 (dichloroacetate 1,10-decandiricinoleate) was obtained at94% yield (1.07 g).

FT-IR ν_(max) 3022, 2930, 2922, 2855, 1759 (COCH₂Cl), 1737, 1453, 1426,1371, 1291 (CH₂Cl), 1264, 1185, 1083, 1015, 964, 849, 719 cm-1.

Precursor 17 (dichloroacetate 1,2-ethanmonoricinoleate) was obtained at92% yield (0.47 g).

FT-IR ν_(max) 3014, 2962, 2927, 2856, 1759 (COCH₂Cl), 1740, 1468, 1416,1384, 1309, 1291 (CH₂Cl), 1254, 1172, 1037, 964, 863, 778 cm⁻¹.

Precursor 18 (dichloroacetate 1,10-decanmonoricinoleate) was obtained at94% yield (0.8 g).

FT-IR ν_(max) 3011, 2928, 2856, 1760, 1736, 1472, 1286, 1257, 1175,1009, 787 cm⁻¹.

[α]²⁰ ₅₈₉=+14.7 (solvent: CHCl₃; 0.1313 g/ml)

v. Synthesis of Derivatives 6-10

A mixture of dichloroacetate Precursors 14-18 (1 mole) andN,N-dimethylaminoethyl acetate (8 moles) was stirred at 75-80° C. for 3hours. After cooling, the reaction mixture was purified by silica gelcolumn chromatography eluted with acetonitrile:water (10:1) or washedseveral times with CH₂Cl₂ and diethyl ether to remove excess ofN,N-dimethylaminoethylacetate and pure amphiphilic products wereobtained.

Derivative 6 was obtained at 50% yield after CC (130 mg)

FT-IR ν_(max) 3007, 2950, 2923, 2854, 1743, 1645, 1471, 1367, 1238,1158, 1021, 876 cm-1.

¹H NMR (CDCl₃, 500 MHz) δ ppm 5.52 (2H, m, CH₂—CH═CH—CH₂—CH(O)), 5.31(2H, m, CH₂—CH═CH—CH₂—CH(O)), 4.95 (2H, m, CH═CH—CH₂—CH(O)), 4.95, 4.87(4H, 2d, 17.0 Hz, O—CO—CH ₂—N⁺), 4.56 (4H, m, N⁺—CH₂CH ₂—O), 4.34 (4H,m, N⁺—CH ₂CH₂—O), 3.73, 3.73 (12H, 2s, CH ₃—N⁺—CH ₃), 4.27 (4H, s,CH₂—CH ₂—O—CO), 2.32 (8H, 2t, 7.5 Hz, O—CO—CH ₂—CH₂, CH═CH—CH ₂—CH(O)),2.02 (4H, m, CH ₂—CH═CH—CH₂—CH(O)), 2.10 (6H, s, CH ₃—CO), 1.60, 1.30(40H, m, CH ₂), 0.88 (6H, t, 6.5 Hz, CH ₃—CH₂). ¹³C NMR (CDCl₃, 500 MHz)δ ppm 173.57 (CO—CH₂—CH₂), 169.81 (CO—CH₃), 164.52 (CO—CH₂—N⁺), 133.60(CH₂—CH═CH—CH₂—CH(O)), 123.22 (CH₂—CH═CH—CH₂—CH(O)), 62.69(CH═CH—CH₂—CH(O)), 62.21 (O—CO—CH₂—N⁺), 57.73 (N⁺—CH₂ CH₂—O), 62.21(N⁺—CH₂CH₂—O), 52.62 (CH₃—N⁺—CH₃), 62.69 (CH₂—CH₂—O—CO), 33.29(O—CO—CH₂—CH₂), 34.15 (CH═CH—CH₂—CH(O)), 27.38 (CH₂—CH═CH—CH₂—CH(O)),20.83 (CH₃—CO), 31.59, 29.40, 29.07, 25.26, 24.86, 22.56 (CH₂), 14.03(CH₃—CH₂). MS (ESI) m/z: 483.66 [(M-2Cl⁻+H⁺)/2], 1037.32 Calcd forC₅₄H₉₈O₁₂Cl₂ N₂.

Derivative 7 was obtained at 67% yield after CC (486.5 mg).

FT-IR ν_(max) 3018, 2930, 2856, 1740, 1650, 1462, 1387, 1238, 1208,1155, 1065, 943, 872, 794 cm-1.

¹H NMR (CDCl₃, 500 MHz) δ ppm 5.50 (2H, m, CH₂—CH═CH—CH₂—CH(O)), 5.30(2H, m, CH₂—CH═CH—CH₂—CH(O)), 4.95 (2H, m, CH═CH—CH₂—CH(O)), 4.88, 4.84(4H, 2d, 17 Hz, O—CO—CH ₂—N⁺), 4.58 (4H, m, N⁺—CH₂CH ₂—O), 4.35 (4H, m,N⁺—CH ₂CH₂—O), 3.73 (12H, s, CH ₃—N⁺—CH ₃), 4.09 (4H, 2t, 5.0 Hz, CH₂—CH₂—O—CO), 2.34 (4H, m, O—CO—CH ₂—CH₂) 2.29 (4H, t, 7.5 Hz, CH═CH—CH₂—CH(O)), 2.02 (4H, q, 5.5 Hz, CH ₂—CH═CH—CH₂—CH(O)), 2.10 (6H, s, CH₃—CO), 1.70, 1.60, 1.30 (44H, m, CH ₂), 0.88 (6H, t, 7.0 Hz, CH₃—CH₂).). ¹³C NMR (CDCl₃, 500 MHz) δ ppm 173.84 (CO—CH₂—CH₂), 169.80(CO—CH₃), 164.50 (CO—CH₂—N⁺), 133.61 (CH₂—CH═CH—CH₂—CH(O)), 123.20(CH₂—CH═CH—CH₂—CH(O)), 63.73 (CH═CH—CH₂—CH(O)), 62.64 (O—CO—CH₂—N⁺),57.80 (N⁺—CH₂ CH₂—O), 62.64 (N⁺—CH₂CH₂—O), 52.67 (CH₃—N⁺—CH₃), 63.73(CH₂—CH₂—O—CO), 34.30 (CH═CH—CH₂—CH(O)), 33.27 (O—CO—CH₂—CH₂), 27.37(CH₂—CH═CH—CH₂—CH(O)), 20.85 (CH₃—CO), 31.59, 29.38, 29.04, 25.35,25.25, 24.92, 22.56 (CH₂), 14.03 (CH₃—CH₂). MS (ESI) m/z: 497.62[(M-2Cl⁻)/2], 1066.24 calcd for C₅₆H₁₀₂O₁₂Cl₂N₂. Argentometric titrationcalcd: Cl⁻ 6.67%. found: 6.41%.

Derivative 8 was obtained at 65% yield after washing (220.9 mg).

FT-IR (NaCl) ν_(max) 3016, 2926, 2856, 1741, 1638, 1460, 1427, 1379,1236, 1172, 1038, 946 cm-1.

¹H NMR (CDCl₃, 500 MHz) δ ppm 5.53 (2H, m, CH₂—CH═CH—CH₂—CH(O)), 5.30(2H, m, CH₂—CH═CH—CH₂—CH(O)), 4.96 (2H, m, CH═CH—CH₂—CH(O)), 4.91, 4.83(4H, 2d, 17.0 Hz, O—CO—CH ₂—N⁺), 4.59 (4H, m, N⁺—CH₂CH ₂—O), 4.37 (4H,m, N⁺—CH ₂CH₂—O), 3.75 (12H, s, CH ₃—N⁺—CH ₃), 4.07 (4H, t, 6.5 Hz,CH₂—CH ₂—O—CO), 2.36 (4H, m, O—CO—CH ₂—CH₂), 2.31 (4H, t, 7.5 Hz,CH═CH—CH ₂—CH(O)), 2.03 (4H, q, 7.0 Hz, CH ₂—CH═CH—CH₂—CH(O)), 2.12 (6H,s, CH ₃—CO), 1.62, 1.32 (56H, m, CH ₂), 0.89 (6H, t, 7.0 Hz, CH₃—CH₂).). ¹³C NMR (CDCl₃, 500 MHz) δ ppm 173.83 (CO—CH₂—CH₂), 169.87(CO—CH₃), 164.11 (CO—CH₂—N⁺), 133.86 (CH₂—CH═CH—CH₂—CH(O)), 123.16(CH₂—CH═CH—CH₂—CH(O)), 64.36 (CH═CH—CH₂—CH(O)), 62.69 (O—CO—CH₂—N⁺),57.73 (N⁺—CH₂ CH₂—O), 62.69 (N⁺—CH₂CH₂—O), 52.68 (CH₃—N⁺—CH₃), 64.36(CH₂—CH₂—O—CO), 34.36 (CH═CH—CH₂—CH(O)), 33.27 (O—CO—CH₂—CH₂), 27.37(CH₂—CH═CH—CH₂—CH(O)), 20.81 (CH₃—CO), 31.58, 29.38, 39.33, 29.10,29.05, 28.60, 25.88, 25.24, 24.96, 22.55 (CH₂), 14.01 (CH₃—CH₂). MS(ESI) m/z: 539.7 [(M-2Cl⁻)/2], 1150.4 calcd for C₆₂H₁₁₄O₁₂Cl₂ N₂.Argentometric titration calcd: Cl⁻ 6.18%. found: 6.17%.

Asymmetric Derivative 9 was obtained at 37% yield (220 mg).

FT-IR ν_(max) 3011, 2930, 2856, 1756, 1743, 1667, 1456, 1378, 1238,1204, 1160, 1090 cm-1.

Asymmetric Derivative 10 was obtained at 47% yield (330 mg).

FT-IR ν_(max) 3031, 2926, 2855, 1746, 1652, 1467, 1376, 1239, 1059, 754cm⁻¹.

¹H NMR (CDCl₃, 500 MHz) δ ppm 5.50 (1H, m, CH₂—CH═CH—CH₂—CH(O)), 5.30(1H, m, CH₂—CH═CH—CH₂—CH(O)), 4.98 (2H, s, CH₂—CH₂—O—CO—CH ₂—N⁺), 4.94(1H, m, CH═CH—CH₂—CH(O)—CO), 4.79 (2H, s, CH—O—CO—CH ₂—N⁺), 4.59, (2H,m, CH—O—CO—N⁺—CH₂CH ₂—O), 4.55 (2H, t, 5.0 Hz, CH₂—O—CO—N⁺—CH₂CH ₂—O),4.31 (2H, m, CH—O—CO—N⁺—CH ₂CH₂—O), 4.28 (2H, m, CH₂—O—CO—N⁺—CH₂CH ₂—O),4.17 (2H, t, 7.0 Hz, CH₂—CH ₂—O—CO—CH₂N⁺), 4.06 (2H, t, 6.5 Hz, CH₂—CH₂—O—CO—CH₂), 2.88 (12H, s, CH ₃—N⁺—CH ₃), 2.34 (2H, m, O—CO—CH ₂—CH₂, CH₂), 2.29 (2H, t, 7.5 Hz, CH═CH—CH ₂—CH(O)), 2.09, 2.07 (6H, 2s, CH₃—CO),2.03 (2H, q, 7.5 Hz, CH ₂—CH═CH—CH₂—CH(O)), 1.62, 1.30 (26H, m, CH ₂),0.87 (3H, t, 7.0 Hz, CH ₃—CH₂). ¹³C NMR (CDCl₃, 500 MHz) δ ppm 173.88(CO—CH₂—CH₂), 170.23, 169.788 (CO—CH₃), 165.09, 164.49 (CO—CH₂N⁺),133.58 (CH₂—CH═CH—CH₂—CH(O)), 123.21 (CH₂—CH═CH—CH₂—CH(O)), 66.71(CH═CH—CH₂—CH(O)—CO), 64.25 (CH₂—CH₂—O—CO—CH₂N⁺), 62.62(CH—O—CO—CH₂—N⁺), 62 (CH₂—CH₂—O—CO—CH₂—N⁺), 58.07(CH(O)CO—CH₂—N⁺—CH₂—CH₂—O), 57.84 (CH₂—O—CO—CH₂—N⁺—CH₂—CH₂—O), 62(N⁺—CH₂CH₂—O), 52.79 (CH₃—N⁺—CH₃), 62.73 (CH₂—CH₂—O—CO—CH₂), 34.41(CH═CH—CH₂—CH(O)), 33.29 (O—CO—CH₂—CH₂), 27.35 (CH₂—CH═CH—CH₂—CH(O)),31.57, 30.82, 29.34, 29.25, 29.13, 29.01, 28.57, 28.25, 25.85, 25.60,25.22, 24.99, 22.52 (CH₂), 13.96 (CH₃—CH₂). MS (ESI) m/z: 399.54[(M-2Cl⁻)/2], 870.08 Calcd for C₄₄H₈₂O₁₀Cl₂ N₂.

Example 9 Preparation of Derivative 4-Nanoparticles Loaded with LeuEnkephalin

Derivative 4, presented in Scheme 5, was synthesized as described inGrinberg et al. (Grinberg et al., 2008). This bolaamphiphile, bearingtwo acetylcholine headgroups, wherein the acetyl choline is linked tothe amphiphile via its nitrogen atom, was used for the preparation ofnanoparticles comprising leu-enkephalin. The additives cholesterol andcholesteryl hemisuccinate were added to Derivative 4 in order to confermore stability to the vesicles which form from the symmetricbolaamphiphile.

The following stock solutions were prepared: 10 mg/kg leu-enkephalin inTBS (TBS), pH 8.5; Derivative 4 (10 mg/ml); dissolved in chloroformtogether with the additives cholesteryl hemisuccinate and cholesterol(1:1). From these stock solutions, a thin film was prepared and hydratedin the following procedure:

Derivative 4, cholesterol and cholesteryl hemisuccinate in molar ratioof 100:25:25, respectively, mixed in chloroform were placed in a roundbottom flask attached to a vacuum evaporator. Evaporation took place for2 hours under vacuum to obtain a dry film.

One (1) ml of the leu-enkephalin solution was added into the roundbottom flask containing the dry film, and the film was hydrated for 20min while rotating the flask (without vacuum) to obtain a suspension.This suspension was extruded through a membrane 100 nm microporous(Nucleopore) until the solution becomes transparent (approx. 8-10times).

The leu-enkephalin was encapsulated at a pH above its pI point and thushad a net negative charge, which interacted with the oppositely chargedheadgroups of Derivative 4. The percentage of encapsulation was to 2 to4 times higher than when leu-enkephalin was encapsulated at a pH belowits pI point, where it had a net cationic charge.

Example 10 Preparation of Nano-Sized Particles Comprising DistearylPhosphatidyl Choline and Leu Enkephalin

Nanoparticles comprising distearyl phosphatidyl choline (DSPC) as theamphiphilic compound and cholesterol as an additive where made by theFHE technique as described in Example 9, at 65° C., which is above thetransition point of DSPC.

Example 11 Preparation of Nano-Sized Particles Comprising Derivative 1or Derivative 4 and Leu Enkephalin

Nanoparticles comprising leu-enkephalin encapsulated within vesiclesmade form Derivative 1 or Derivative 4 were prepared by the ethanolinjection technique. The bolaamphiphiles Derivative 1 and 4 have asimilar amphiphilic backbones but the acetyl choline headgroups[CH₃—C(O)—O—(CH₂)₂—N⁺(CH₃)₃] are bonded to the backbone differently: viathe quaternary nitrogen in Derivative 4 (see Scheme 5) and via themethyl group in Derivative 1 (see Scheme 4).

An ethanolic solution of Derivative 1 or Derivative 4 (10 mgbolaamphiphile in 50 μl ethanol) was prepared and injected into a 1 mlstirring aqueous solution (saline, 0.9% NaCl) containing 2.5 mgleu-enkephalin. The resulting suspension was sonicated in a bathsonicator (36 KHz) at 35° C. for 1 hour to form nanoparticles of about100 nm in diameter. These nanoparticles were used (administered to mice)no later than 1 hour after preparation thereof.

Example 12 Preparation of Nano-Sized Particles Comprising Derivative 2or Derivative 3

Asymmetric bolaamphiphiles Derivatives 2 and 3 were synthesized asdescribed in Examples 5 and 6, respectively, and nanoparticles based onthese amphiphiles were prepared by the ethanol injection technique, asfollows:

An ethanolic solution of Derivative 2 or Derivative 3 (10 mgbolaamphiphile in 50 μl ethanol) was injected into a stirring aqueoussolution of PBS 0.9% NaCl. The resulting suspension was sonicated in abath sonicator (36 KHz) at 35° C. for 1 hour to form opalescentsolutions.

Examination of the solutions under transmission electron microscopy(TEM) showed that Derivative 2 formed vesicles and ribbons whileDerivative 3 formed primarily nano sized vesicles.

The stability of nano vesicles formed from the asymmetric Derivative 3was studied by dynamic light scattering (DLS) and TEM and compared tothe stability of vesicles made from the symmetric bolaamphiphileDerivative 4 by exactly the same procedure. Suspensions containing thevesicles were left to stand for 1, 5, 10 and 30 days to assess thestability of the nanoparticle solution/suspension, and then analyzed byDLS and TEM. The results showed that vesicles made from asymmetricDerivative 3 were considerably more stable with respect to size andhomogeneity.

Example 13 Preparation of Nano-Sized Particles Comprising the AdditiveVernoyl Chitosan

Nanoparticles comprising various active agents encapsulated withinvesicles made from various amphiphiles along with the additive vernolylchitosan were prepared by the thin film technique as described inExample 9. Nanoparticles comprising chitosan additive are most suitablefor the preparation of oral formulations.

i. Nanoparticles Comprising Derivative 4 Encapsulating Leu-Enkephalin

Solutions of 10 mg/kg leu-enkephalin in TBS pH 8.5, 10 mg/ml Derivative4 in 300 μl chloroform, was mixed with the additives cholesterylhemisuccinate and cholesterol (1:1) each separately dissolved in 300 μlof chloroform to form a solution with a molar ratio of 100:25:25 forDerivative 4, cholesterol and cholesteryl hemisuccinate, respectively.The solution was evaporated under for 2 hours under vacuum to obtain adry film. One (1) ml solution of 2.5 mg leu-enkephalin were added, andthe film was hydrated for 20 min to obtain a suspension.

The suspension was transferred into a 5 ml glass vial, and 10 molarpercent (or 1 mg) of vernolyl chitosan (MW ˜80K) obtained according toExample 1 were added (the molar ratio of Derivative4:cholesterol:cholesteryl hemisuccinate:chitosan was 100:25:25:10,respectively). Dissolution of chitosan in the bolaamphiphile-activeagent reaction mixture simultaneously with its incorporation into thevesicles membrane and vesicle formation was carried out in the samesolution by sonication (probe sonication: sonicate 30% power, pulses 10seconds, rest 10 seconds for 15 minutes until complete. The chitosanappeared to dissolve as indicated by the disappearance of turbidity).

ii. Nanoparticles Comprising Derivative 4 EncapsulatingCarboxyfluorescein

The marker carboxyfluorescein (CF) was used as a model of non polymericactive agents. Nanoparticles comprising Derivative4/cholesterol/cholesteryl hemisuccinate/vernoyl chitosan (molar ratio100:25:25:10, respectively) encapsulating CF, were prepared as describedabove using a solution of 1 mg/ml CF for hydration of the thin film.

iii. Nanoparticles Comprising Distearyl Phosphatidyl CholineEncapsulating Leu-Enkephalin

Nanoparticles comprising distearyl phosphatidyl choline (DSPC), andcholesterol (molar ratio 100:30, respectively) encapsulatingleu-enkephalin, were prepared as described in (i) but the amount of DSPCdissolved in chloroform was 20 mg instead of 10 mg and twice as that ofDerivative 4.

Example 14 Preparation of Nano-Sized Particles Comprising a Mixture ofBolaamphiphiles

For the purpose of optimizing the delivery efficiency, active agenttargeted release efficiency, stability and durability in the bloodstream, nanoparticles comprising mixtures of different bolaamphiphileswere prepared.

(i) Nanoparticles comprising Derivative 4, Derivative 5 andLeu-Enkephalin

Nanoparticles comprising a mixture of the symmetric bolaamphiphilesDerivative 4 and Derivative 5 and encapsulated leu-enkephalin, wereprepared according to the procedure described in Example 9, startingwith a solution comprising these two derivatives in a weight ratio of9:1 Derivative 4:Derivative 5, respectively dissolved in chloroform.

(ii) Nanoparticles comprising Derivative 1, Derivative 4 andLeu-Enkephalin

Nanoparticles comprising a mixture of the symmetric bolaamphiphilesDerivative 1 and Derivative 4 and leu-enkephalin, and vernoyl chitosanas additive were prepared according to the procedure described inExample 13, starting with a solution comprising these two derivatives ina weight ratio of 2:1 Derivative 1:Derivative 4, respectively dissolvedin chloroform.

Example 15 Preparation of Nano-Sized Particles Comprising Derivative 4,Vernoyl Chitosan and Various Amounts of Leu Enkephalin

For the purpose of optimizing the nanoparticles' packing, deliveryefficiency, stability, or surface properties, nanoparticles wereprepared form Derivative 4 and vernoyl chitosan as additive, and variousamounts of leu-enkephalin. Nanoparticles were prepared as described inExample 13 using solutions of 5, 10 or 20 mg/kg leu-enkephalin in TBS pH8.5. Instead of extrusion through a filter the solution containing thenanoparticles was probed sonicated at RT until the temperature raised to40° C.

Example 16 Preparation of Nano-Sized Particles Comprising Derivative 4,Carboxyfluorescein and PEG-Vernonia Derivatives

Nanoparticles comprising the fluorescent marker carboxyfluorescein (CF)encapsulated within vesicles made from Derivative 4 and decorated withPEG₂₀₀₀-vernonia derivatives, cholesteryl hemisuccinate and cholesterolas pendants were prepared by the thin film technique as described inMethods and in Example 9. Two kinds of PEG-vernonia derivatives wereused: PEG-ether derivatives, wherein PEG is bound via an ether bond tothe oxygen of the opened epoxy ring of vernolic acid (PEG(202)), andPEG-ester derivatives, wherein PEG is bound via an ester bond to thecarboxylic group of vernolic acid (PEG(201)). These PEG-vernoniaderivatives were prepared as described in Example 3.

Solutions of 20 mg/ml CF in PBS pH 8.0, 10 mg/ml Derivative 4 in asolution of the additives cholesteryl hemisuccinate and cholesterol(1:1) in chloroform were prepared. PEG(201) and PEG(202) were added assolids and dissolved to obtain a solution comprising Derivative 4,cholesterol, cholesteryl hemisuccinate and PEG(201) or PEG(202) in amolar ratio of 100:25:25:10, respectively. A dry film was obtained asdescribed in Example 9. Then, 1 ml of the PBS solution of CF were added,and the film was hydrated for 20 min. The particles were not isolatedand used in the solution they were made.

The percentage of CF encapsulation was 20%. This relatively highencapsulation percentage is attributed to electrostatic interactionsbetween the anionic groups of CF and the cationic headgroups of thebolaamphiphile.

Example 17 Preparation of Nano-Sized Particles Comprising Derivative 4and Ovalbumin

Nanoparticles comprising tritiated ovalbumin encapsulated withinvesicles made from Derivative 4 and the additives cholesterylhemisuccinate and cholesterol were prepared by the FHE technique asdescribed in Methods and in Example 9.

Briefly, the following solutions were prepared: 10 mg/kg tritiatedovalbumin in PBS at a pH 8.0, above its pI point, 10 mg/ml Derivative 4in 300 μl in chloroform, cholesteryl hemisuccinate and cholesterol (1:1)each dissolved in 300 μl chloroform. The solutions of Derivative 4,cholesterol and cholesteryl hemisuccinate were mixed to give a molarratio of 100:25:25, added to a round bottom flask and a dry film wasobtained. The thin film was hydrated for 20 min with 1 ml of theovalbumin solution and the suspension thus obtained was extruded througha membrane 100 nm microporous (Nucleopore) until the solution becomestransparent (approx. 8-10 times).

Example 18 Preparation of Nano-Sized Particles Comprising Derivative 4and ¹²⁵I-GDNF

Nanoparticles comprising ¹²⁵I-GDNF (glial cell line-derived neurotrophicfactor) encapsulated within vesicles made from Derivative 4 and theadditives cholesteryl hemisuccinate and cholesterol are prepared by thethin film technique as described in Example 17, using a stock solutionof 2 mg/kg ¹²⁵I-GDNF in TBS at pH 9.5 for hydrating the thin film andforming nanoparticles encapsulating ¹²⁵I-GDNF.

Example 19 Preparation of Nano-Sized Particles Comprising Derivative 1and a DNA Plasmid

Nanoparticles comprising Derivative 1 and a DNA plasmid were prepared bythe FHS technique as follows:

10 mg of Derivative 1 was prepared by dissolving it in 1 ml chloroformin a 50 ml round-bottom flask. The solvent was removed under reducedpressure and the thin film so obtained was dried overnight in a vacuumdesiccator to remove traces of solvent. To this dried film, 1 ml ofphosphate-buffered saline containing 0.1 mg of the BGFP-N1 reporter geneencoding a red-shift variant of the wild-type green fluorescent protein(GFP) was added. The mixture was then sonicated to form nanoparticlesencapsulating the DNA.

Biological Section Materials

Mice. Eight weeks old male ICR mice, weighing between 25-30 g, weremaintained on a standard mice chow and tap water ad lib. The mice werekept in a 12 hours light/dark cycles with temperature of 25±3° C. Allanimals were handled and tested according to an approved protocol (#IL-24-04-2008).

Cells. COS-7 cells, used as target cells for transfection, were grown in96-well plates or in 30-mm petri dishes to 40-50% confluence.

Methods

i. Transfection. Transfection with DEAE-dextran was used both as apositive control and as a reference method. Transfection efficiency wasdetermined by counting the number of transfected cells (greenfluorescent cells) per total number of cells seen in the same field by afluorescent microscope.ii. Determination of Analgesic Effect (Hot Plate Test)

The response of mice to a transient painful stimulus was measuredfollowing administration of the test material (either i.v. into the tailvein or per os by gavages). The analgesic effect was determined byplacing the mouse on a hot plate (55° C., IITC model 39) and recordingthe time for withdrawing/licking the hind limb (hot plate test). Toprevent tissue damage and suffering of the animal, the experiment wasterminated after 20 sec if no response was evoked. The responselatencies were recorded and either used by themselves for comparison ornormalized as percent of maximal possible effect (MPE) using theequation:

${\% \mspace{14mu} M\; P\; E} = {\frac{\left( {{RT} - {RT}_{0}} \right)}{\left( {{RT}_{\max} - {RT}_{0}} \right)} \cdot 100}$

where: RT—is the response latency after treatment; RT₀—is the responselatency of a mouse without treatment and RT_(max)—is the maximalresponse time allowed (20 sec).

iii. Tissue Distribution of Carboxyfluorescein (CF)

For tissue distribution of CF, tissue specimens dissected out from micethat were sacrificed 30 min after injection of nanoparticles loaded withCF, were weighed and homogenized in PBS at a dilution of 1:4 (w/w tissueto PBS). TCA (10%) was added to the homogenates at a ratio of 1:1 toachieve a final concentration of 5% TCA. The specimens were transferredto Eppendorf tubes and centrifuged for 5 minutes at 13,200 rpm. NaOH wasadded to neutralize the acid (the volume of the NaOH was predeterminedby titrating 5% TCA until a pH 7.0 was obtained). The supernatants wereused for the fluorimetric determinations at an excitation wavelength of492 nm, using the scan program.

iv. Brain Uptake of Fluorescein Isothiocyanate (FITC)-Albumin

The amount of FITC-albumin in the brain was measured using the methodfor assessing the distribution of CF.

Example 20 Analgesic Effect in Mice Treated with NanoparticlesComprising Leu-Enkephalin

Derivative 4-nanoparticles comprising leu-enkephalin, prepared asdescribed in Example 9 by FHS (film hydration flowed by sonication),were administered to mice and their ability to exert an analgesic effectwas measured by the hot plate test described in Methods.

Nanoparticles were made from 10 mg/ml Derivative 4 with cholesterol andcholesteryl hemisuccinate as additives (2:1:1) and 10% molar ratiochitosan (CS)-vernolic acid conjugate in presence of 2.5 mg/mlleu-enkephalin. In these nanoparticles, CS-vernolic acid conjugateserves as a pendant on the nanoparticles/vesicles in order to enhancepenetrability.

Nanoparticles were injected into the tail vein at a dose of 20 mg/kgbolaamphiphile, which corresponds to 5 mg/kg leu-enkephaime. Emptynanoparticles and free leu-enkephalin injected in a dose of 20 mg/kg,were used as a negative control and morphine at a dose of 5 mg/kg wasused as a positive control. Mice were pretreated with 0.5 mg/kgpyridostigmine 15 min prior to the injection of the nanoparticles.Pyridostigmine inhibits the activity of acetylcholine esterase and thusprevents hydrolysis of the acetyl choline headgroups in the periphery.The drug does not penetrate into the CNS and cannot prevent hydrolysisof the acetylcholine headgroups in the CNS. By itself, pyridostigminedoes not evoke an analgesic response.

The results shown on FIG. 1 represent the effect as percent of themaximal possible effect (MPE).

As shown in FIG. 1, nanoparticles comprising leu-enkephalin induced aresponse which was 3 to 8 times greater than free enkephalin (dependingon the time after injection), and approached the efficacy of morphine 30min after injection. At 60 and 90 minutes, encapsulated leu-enkephalinwas more efficient than morphine.

Empty nanoparticles without leu-enkephalin had no effect beyond thecontrol. When the nanoparticles with leu-enkephalin were checked withouta pre-injection of pyridostigmine they were about 3 to 7 times lesseffective than with pre-injection of pyridostigmine (not shown).

Example 21 Analgesic Effect in Mice i.v. Treated with NanoparticlesComprising Leu-Enkephalin

Analgesic effect studies (hot plate test) in mice were carried out usingnanoparticles comprising Derivative 1 or Derivative 4 and prepare by theethanol injection technique described in Example 10. The hot plate testwas preformed as described in Example 19. All mice treated withencapsulated leu-enkephalin, were pre-injected with the peripheryacetylcholine esterase inhibitor pyridostigmine (0.5 mg/kg).

The response time (in seconds) was checked 10, 30 and 60 minutes afterinjection and showed for free leu-enkephalin a response time of 3, 2 and2 sec respectively, while the formulation with leu-enkephalin innanoparticles of Derivative 1 showed a response time of 5, 5 and 5 secrespectively, and the formulation of leu-enkephalin encapsulated inDerivative 4-nanoparticles showed a response time of 11, 13 and 6 secrespectively. The results demonstrate that leu-enkephalin encapsulatedin Derivative 4 nanoparticles had a significant analgesic effectcompared to non-encapsulated leu enkephalin. These results also showthat the efficacy of nanoparticles comprising Derivative 4 prepared bythe ethanol injection technique to deliver enkephalin into the CNS isthe same as that of corresponding nanoparticles prepared by the thinfilm technique. In addition, nanoparticles comprising Derivative 1 areclearly less efficient than those comprising Derivative 4, but stillmore efficient compared to non-encapsulated enkephalin.

Example 22 Analgesic Effect in Mice Orally Treated with NanoparticlesComprising Leu-Enkephalin

The efficacy of delivering leu-enkephalin in various nanoparticles inoral formulations versus i.v. injectable formulations were tested (a hotplate test of Example 20). Thus, formulations comprising leu-enkephalinencapsulated in the following nanoparticles were prepared: (a)nanoparticles comprising Derivative 4, cholesterol, cholesterylhemisuccinate and vernolyl-chitosan prepared according to Example 13(i);(b) nanoparticles comprising distearyl phosphatidyl choline (DSPC) asthe amphiphilic compound, cholesterol, prepared as described in Example13(iii).

Formulations comprising free leu-enkephalin loaded nanoparticles wereadministered by gavages at a dose of 100 mg/kg bolaamphiphile, whichcorresponds to 25 mg/kg leu-enkephalin. The same dose of freeleu-enkephalin was administered by gavages as a control. The resultswere compared to liposomes made from 20 mg/ml DSPC in presence of 5mg/ml leu-enkephalin and administered at a dose of 200 mg/kgphospholipid, which corresponds to 50 mg/kg leu-enkephalin. The micewere preinjected with pyridostigmine as in example 20. The results, areshown in FIG. 2, represent the analgesic effect as percent of maximalpossible response (MPE).

The results show that formulations of leu-enkephalin encapsulated inDerivative 4-nanoparticles showed a significantly stronger effect thanfree enkephalin by a factor of more than 3. The analgesic effect of theoral DSPC liposome was similar to the control of three leu-enkephalinand two to three times less than the Derivative 4-nanoparticles.

Example 23 Brain Uptake of Carboxyfluorescein Delivered in NanoparticlesComprising Derivative 4 and PEG₂₀₀₀-Vernonia Derivatives

The ability of nanoparticles of the invention to penetrate into thebrain tissue was assessed by using the fluorescent marker CFencapsulated in nanoparticles comprising the bolaamphiphile Derivative4, a PEG₂₀₀₀-vernonia derivative (PEG(202)) and the additivescholesteryl hemisuccinate. The nanoparticles were prepared as describedin Example 16.

Two group of mice (2 mice in each) were pre-injected with pyridostigmine(0.5 mg/kg), and then treated as follows: group (i) was i.v. injectedwith free (non-encapsulated) CF (0.333 mg/Kg); group (ii) was i.v.injected with a formulation containing Derivative4/cholesterol/cholesteryl hemisuccinate/PEG(202) nanoparticles (10mg/kg) loaded with CF (0.333 mg/Kg). Brain uptake was determined after30 minutes as described in Method, and the results are shown in FIG. 3.

As seen in FIG. 3, the formulation containing nanoparticles comprisingPEG(202) as pedant or additive was taken up by the brain 6 time morethan free CF, and twice as much as the formulation containingnanoparticles comprising the ester derivative PEG(201) as pendant (thislast comparison is not shown in FIG. 3). This experiment shows that byusing the nanoparticles of the invention, low molecular compounds whichdo not normally enter or distribute into certain organs may neverthelessbe delivered to such organs. This demonstrates the potential of thepresent invention as a delivery system for non polymeric/macromolecularmaterials

Example 24 Brain Uptake of Ovalbumin Delivered in NanoparticlesComprising Derivative 4

Nanoparticles comprising FITC-albumin prepared as described in Example17 are administered to mice and their ability to be taken up by thebrain was measured.

Two groups of mice (3 mice in each) are treated as follows: group (i) isi.v. injected with free (non-encapsulated) FITC-albumin (30 mg/Kg);group (ii) is i.v. injected with a formulation containing Derivative4/cholesterol/cholesteryl hemisuccinate nanoparticles (10 mg/kg) loadedwith ovalbumin (30 mg/Kg). The mice are sacrificed after 30 minutes andbrain uptake is determined, by well known flouremetric techniques forFITC conjugates (see Methods section).

The results are expected to show a significantly greater uptake in thebrain of the encapsulated oval albumin as compared to the free ovalalbumin.

Example 25 Brain Uptake of ¹²⁵I-GDNF Delivered in NanoparticlesComprising Derivative 4

Nanoparticles comprising ¹²⁵I-GDNF prepared as described in Example 18are administered to mice and their ability to be taken up by the brainis measured.

Two groups of mice (3 mice in each) are treated as follows: group (i) isi.v. injected with free (non-encapsulated) ¹²⁵I-GDNF (10 mg/Kg); group(ii) is i.v. injected with a formulation containing Derivative4/cholesterol/cholesteryl hemisuccinate nanoparticles (10 mg/kg) loadedwith ovalbumin (10 mg/Kg). The mice are sacrificed after 30 minutes andbrain uptake is determined as for CF (see Methods).

The results are expected to show a significantly greater uptake in thebrain of the encapsulated ¹²⁵I-GDNF as compared to the free ¹²⁵I-GDNF.

Example 26 Biodistribution of Carboxyfluorescein Delivered in Nano-SizedParticles

The organ distribution following i.v. injection or oral administrationof CF encapsulated in Derivative 4/cholesterol/cholesteryl hemisuccinatenanoparticles, herein also referred to as “basic nanoparticles” wasdetermined and compared to biodistribution of CF encapsulated inDerivative 4/cholesterol/cholesteryl hemisuccinate/vernolyl-chitosannanoparticles, herein also referred to as “chitosan-nanoparticles”(prepared according to Example 12(ii)). Biodistribution of encapsulatedCF was further assessed relative to i.v. or oral administration of freeCF.

For biodistribution study following i.v. injection of free andencapsulated CF, three groups of mice (5-7 mice per group) were treatedas follows: group (i) was i.v. injected with a formulation containingthe basic nanoparticles (10 mg/kg) loaded with 0.2 mg/ml CF; group (ii)was i.v. injected with a formulation containing thechitosan-nanoparticles (10 mg/kg) loaded with 0.2 mg/ml CF; group (iii)was pre-injected with the cholinesterase inhibitor pyridostigmine (0.5mg/kg) and then i.v. injected with chitosan-nanoparticles loaded with0.2 mg/ml CF.

Mice were sacrificed 30 minutes after i.v. administration and theirbrain, lungs, kidneys, muscle, heart and liver were dissected out. Theorgans were then homogenized, diluted, deproteinated withtrichloroacetic acid (TCA), brought to basic pH, and fluorescenceintensity was measured. The results are shown in FIGS. 4A-4D.

As shown in FIG. 4A, enhanced uptake of CF delivered inchitosan-nanoparticles in mice pre-treated with pyridostigmine (group(iii)) was detected in the targeted organ the brain and also in organswhich contain high levels of AChE enzyme, namely, heart (FIG. 4B) andmuscle (FIG. 4D). In the lungs (which also served used as a control) thelevel of CF remained low (FIG. 4C).

The selective uptake by the brain, heart and muscles following i.v.administration proves that these and similar nanoparticles can be usedfor delivery of therapeutic and preventive agents to these organs,particularly the brain. The active agents may be peptides, proteins,polynucleotides and non polymeric compounds, such as antibacterialagents or agents that control bacterial growth and spreading.

In order to asses the organ distribution of orally administered free andencapsulated CF, three groups of mice (5-7 mice per group) were treatedas follows: group (i) was gavaged (force-fed) with a formulationcontaining basic nanoparticles (10 mg/kg) loaded with 0.2 mg/ml CF;group (ii) was gavaged with a formulation containingchitosan-nanoparticles (10 mg/kg) loaded with 0.2 mg/ml CF; and group(iii) was pre-treated with pyridostigmine (0.5 mg/kg)) and then gavagedwith chitosan-nanoparticles loaded with 0.2 mg/ml CF.

Mice were sacrificed 30 minutes after oral administration and theirbrain, lungs, kidneys, muscle, heart and liver were dissected andanalyzed as described above. The results are shown in FIGS. 5A-5D.

Example 27 Analgesic Effect in Mice Treated with NanoparticlesComprising Various Concentrations of Leu-Enkephalin

In order to assess an optimized delivery and targeted releaseefficiencies, the analgesic effect of formulations containingnanoparticles comprising Derivative 4, cholesterol, cholesterylhemisuccinate and vernoyl chitosan as additives (chitosannanoparticles), and various amounts of leu-enkephalin was studied by thehot plate test. Nanoparticles were prepared as described in Example 15.

Five groups of mice (5 mice in each group) were treated as follows:group (i) i.v. administration of morphine (5 mg/kg). This groups servedas the positive control; group (ii) i.v. administration of chitosannanoparticles (20 mg/kg) loaded with 20 mg/kg leu-enkephalin; group(iii) i.v. administration of chitosan nanoparticles (20 mg/kg) loadedwith 10 mg/kg leu-enkephalin; group (iv) i.v. administration of chitosannanoparticles (20 mg/kg) loaded with 5 mg/kg leu-enkephalin; group andgroup (v) i.v. injection of free (nonencapsulated) leu-enkephalin (20mg/Kg). Four mice were checked for each time point (10, 30, 60 and 90min) after i.v. administration. The mice in group (ii)-(vi) wherepre-injected with pyridostigmine in PBS at a concentration of 0.5 mg/kg.Mice before i.v. treatment served as control and as zero point. The hotplate test was conducted as described in Example 19. The results forgroups (i)-(iv) and (vi) are shown in FIG. 6.

The results show a greater analgesic effect at lower concentration ofenkephalin, 30 and 60 min after administration. Without being bound to aparticular theory, the lower quantity of enkephalin in the nanoparticlesmay give rise to more stable nanoparticles, probably of smaller size,having improved penetrability and/or improved surface properties. Freeleu-enkephalin and empty nanoparticles injected to mice pretreated withpyridostigmine had no significant analgesic effect.

These results indicate that the chitosan nanoparticle penetrated the BBBand released the encapsulated leu-enkephalin within the brain. Theseresults also demonstrate that optimization of delivery can be assessedby conventional optimization procedures.

Example 28 Analgesic Effect in Mice Treated with NanoparticlesComprising Derivative 1 and Derivative 4

In order to optimize delivery efficiency, active agent targeted releaseefficiency, stability and durability in the blood stream, nanoparticlescomprising a mixture of Derivative 1 and Derivative 4 in a weight ratioof 2:1, respectively, and the additives cholesteryl hemisuccinate,cholesterol and vernoyl chitosan were prepared and loaded withenkephalin as described in Example 13(ii). These nanoparticles, alsotermed herein “Derivative 1+Derivative 4 nanoparticles” were i.v.injected to mice and the analgesic effect of encapsulated versus freeactive agent was assessed by the hot plate test described in Example 20.For comparison, analgesic effect of leu-enkephalin encapsulated incation liposomes prepared from dioleoyl trimethylammonium propane(DOTAP) and cholesterol was measured. These liposomes were prepared asdescribed in Methods.

Five groups of mice (5 mice in each group) were treated as follows:group (i) i.v. administration of morphine (5 mg/kg). This groups servedas the positive control; group (ii) i.v. administration of Derivative1+Derivative 4 nanoparticles (20 mg/kg) loaded with 5 mg/kgleu-enkephalin; group (iii) pre-injected with pyridostigmine in PBS at aconcentration of 0.5 mg/kg and then i.v. administration of Derivative1+Derivative 4 nanoparticles (20 mg/kg) loaded with 5 mg/kgleu-enkephalin; group (iv) i.v. administration of DOTAP liposomes (20mg/kg) loaded with 5 mg/kg leu-enkephalin; group (v) i.v. injection offree (non-encapsulated) leu-enkephalin (20 mg/Kg). Four mice werechecked for each time point (10, 30, 60, 90 and 120 min) after i.v.administration. Mice before i.v. treatment served as control and as zeropoint. The results are shown in FIG. 7.

The results clearly demonstrate that nanoparticles comprising a mixtureof the two related bolaamphiphiles Derivative 1 and Derivative 2,significantly increased the duration of the analgesic affect beyond 60min as compared to nanoparticle comprising only one bolaamphiphile,Derivative 4 (chitosan nanoparticles) described in Example 26). As shownin FIG. 10, at 90 and 120 after administration, the % MPE for theDerivative 1+Derivative 4 nanoparticles with pre-injection ofpyridostigmine (group (iii)) was about 42 and 38, respectively, whereasfor the chitosan nanoparticles the % MPE at 90 min was 20 (see FIG. 7.On the other hand, at 30 min, the chitosan nanoparticles of Example 27had a higher analgesic effect compared to Derivative 1+Derivative 4nanoparticles.

In addition, the Derivative 1+Derivative 4 nanoparticles provided asignificant analgesic effect even without pre-injection ofpyridostigmine. The Derivative 1+Derivative 4 nanoparticles had betterdrug delivery characteristics than cationic liposomes. As expected, freeenkephalin had no significant analgesic effect.

Thus by using nanoparticles comprising one type of bolaamphiphilebearing head groups which are more readily hydrolyzed at the target sitemay provide a strong effect in the short term of 10-30 min, while longerterm effects may be achieved with nanoparticle comprising a mixture ofbolaamphiphiles bearing head groups which are hydrolyzed at differenttimes.

This example shows that by combining different bolaamphiphiles, thedelivery and release of active agents such as peptides proteins andpolynucleotides can be optimized.

Example 29 Transfection of Cells with DNA Encapsulated in Nanoparticles

The transfection of a polynucleotide delivered by nanoparticlecomprising Derivative 1 was assessed.

Nanoparticles comprising Derivative 1 and a BGFP-N1 reporter geneencoding a red-shift variant of the wild-type green fluorescent protein(GFP were prepared according to Example 19 and added to COS-7 cells thatwere grown in 96-well plates or in 30-mm petri dishes to 40-50%confluence. Transfection with DEAE-dextran was used both as a positivecontrol and as a reference method. Transfection efficiency wasdetermined by counting the number of transfected cells (greenfluorescent cells) per total number of cells seen in the same field by afluorescent microscope.

The transfection efficiency was dependent on the concentration of theamphiphilic derivative used for the vesicle formation. When theconcentration of the amphiphilic derivative was increased from 5 to 10mg/ml, the transfection efficiency was almost doubled that of theDEAE-dextran. The higher transfection efficiency with nanoparticles wasalso expressed in terms of the amount of cDNA needed for transfection,i.e., with vesicles less cDNA yielded more transfected cells than did alarger amount of cDNA complexed with DEAE-dextran

This example clearly demonstrates the high potential of thenanoparticles of the invention to enhance transfection ofpolynucleotides such as DNA and RNA.

REFERENCES

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1. A nano-sized particle comprising at least one multi-headedamphiphilic compound, in which at least one headgroup of saidmulti-headed amphiphilic compound is selectively cleavable or contains aselectively cleavable group, and at least one biologically active agent,which is both encapsulated within the nano-particle and non-covalentlyassociated thereto.
 2. The nano-sized particle according to claim 1,wherein the biologically active agent is associated to the nano-particlevia non-covalent interactions selected from ionic and polarinteractions, electrostatic forces, hydrophobic interactions, Van derWaals forces or hydrogen bonds.
 3. The nano-sized particle according toclaim 1, wherein the biologically active agent is: (i) ionicallyassociated with the at least one multi-headed amphiphilic compound orforms a salt complex therewith; (ii) embedded or incorporated in thenano-sized particle matrix; or (iii) embedded or incorporated in thenano-sized particle matrix and further ionically associated with thenano-sized particle or forms a salt complex therewith; and when thenano-sized particle comprises a vesicular structure, the biologicallyactive agent is encapsulated within the core of said vesicle andassociated via one or more non-covalent interactions to the vesicularmembrane on the outer surface and/or the inner surface, optionally aspendant decorating the outer or inner surface, and optionally furtherincorporated into the vesicular membrane.
 4. (canceled)
 5. (canceled) 6.The nano-sized particle according to claim 1, in the form of a vesicleor liposome formed from said at least one multi-headed amphiphiliccompound.
 7. The nano-sized particle according to claim 6, wherein saidvesicle is a monolayer vesicle.
 8. The nano-sized particle according toclaim 6, wherein the biologically active agent is encapsulated withinthe core of said vesicle and associated via one or more non-covalentinteractions to the vesicular membrane on the outer surface and/or theinner surface, optionally as pendant decorating the outer or innersurface, and optionally further incorporated into the vesicularmembrane.
 9. The nano-sized particle according to claim 1, comprising amixture of at least one multi-headed amphiphile and at least onesingle-headed amphiphile.
 10. The nano-sized particle according to claim1, wherein said at least one multi-headed amphiphile is abolaamphiphile.
 11. The nano-sized particle according to claim 1,wherein at least one of the headgroups of said multi-headed amphiphiliccompound serve as substrates to enzymes at a target site in a biologicalenvironment, enhance transport of the nano-sized particle throughbiological barriers and/or stabilize the vesicular structure of thenano-sized particle.
 12. The nano-sized particle according to claim 11,wherein at least one of said headgroups is ionically charged.
 13. Thenano-sized particle according to claim 1, wherein said selectivelycleavable headgroup is cleaved under selective conditions selected fromchemical, physical or biological conditions such as change of pH ortemperature, oxidative or reducing conditions, and/or enzymaticconditions.
 14. The nano-sized particle according to claim 13, whereinsaid selectively cleavable headgroup is cleaved enzymatically in abiological environment, particularly in the brain or blood, bydegradatives enzymes selected from hydrolases, esterases such ascholinesterases (ChE) and acetylcholine esterase (AChE), phosphatases,oxidases, decarboxylases such as L-amino acid decarboxylase (AADC),deaminases and isomerases.
 15. The nano-sized particle according toclaim 11, wherein said headgroup is selected from: (i) choline orthiocholine, O-alkyl, N-alkyl or ester derivatives thereof; (ii)non-aromatic amino acids with functional side chains such as glutamicacid, aspartic acid, lysine or cysteine, or an aromatic amino acid suchas tyrosine, tryptophan, phenylalanine and derivatives thereof such aslevodopa (3,4-dihydroxy-phenylalanine) and p-aminophenylalanine; (iii) apeptide or a peptide derivative that is specifically cleaved by anenzyme at a diseased site selected from enkephalin, N-acetyl-ala-ala, apeptide that constitutes a domain recognized by beta and gammasecretases, and a peptide that is recognized by stromelysins; (iv)saccharides such as glucose, mannose and ascorbic acid; and (v) othercompounds such as nicotine, cytosine, lobeline, polyethylene glycol, acannabinoid, or folic acid.
 16. The nano-sized particle according toclaim 10, wherein said bolaamphiphile is a symmetric or asymmetricbolaamphiphile optionally containing at least one hydrogen-bonding groupselected from —OH, —SH, —NH—, —N⁺H₂—, —NH₂, —N⁺H₃, —NH—CO—, —O—CO—NH—,—NH—CO—NH—, —C═NOH, —C(NH₂)═NOH, —C(NH₂)═NO— and —CO—NH₂, located eitherwithin the selectively cleavable headgroup or within the headgroupcontaining the selectively cleavable group or moiety and/or in closeproximity thereto.
 17. The nano-sized particle according to claim 16,wherein said symmetric or asymmetric bolaamphiphile further contains oneor more branching alkyl chains bearing pendants such as chitosanderivatives, polyamines or certain peptides, which enhance penetrationthrough various biological barriers.
 18. The nano-sized particleaccording to claim 16, wherein said bolaamphiphile comprises at leastone of said hydrogen bonding group, and is selected from the compoundherein designated Derivatives 1 to
 5. 19. The nano-sized particleaccording to claim 16, wherein said bolaamphiphile does not comprisesaid hydrogen bonding group, and is selected from the compound hereindesignated Derivatives 6 to
 10. 20. The nano-sized particle according toclaim 1, further comprising at least one additive for targetingpurposes, enhancing permeability and/or increasing the stability of thenano-sized particle, said additives are selected from: (i) a singleheaded amphiphilic derivative comprising one, two or multiple aliphaticchains, preferably two aliphatic chains linked to a midsection/spacerregion such as —NH—(CH₂)₂—N—(CH₂)₂—N—, or —O—(CH₂)₂—N—(CH₂)₂—O—, and asole headgroup, optionally a selectively cleavable headgroup or onecontaining a polar or ionic selectively cleavable group or moiety,attached to the N atom in the middle of said midsection; (ii)cholesterol and cholesterol derivatives such as cholesterylhemmisuccinate; (iii) phospholipids, zwitterionic, acidic, or cationiclipids; (iv) chitosan and chitosan derivatives, such as vernolicacid-chitosan conjugate, quaternized chitosan, chitosan-polyethyleneglycol (PEG) conjugates, chitosan-polypropylene glycol (PPG) conjugates,chitosan N-conjugated with different amino acids, carboxyalkylatedchitosan, sulfonyl chitosan, carbohydrate-branchedN-(carboxymethylidene) chitosan and N-(carboxymethyl) chitosan; (v)polyamines such as protamine, polylysine or polyarginine; (vi) ligandsof specific receptors at a target site of a biological environment suchas nicotine, cytisine, lobeline, 1-glutamic acid MK801, morphine,enkephalins, benzodiazepines such as diazepam (valium) and librium,dopamine agonists, dopamine antagonists tricyclic antidepressants,muscarinic agonists, muscarinic antagonists, cannabinoids andarachidonyl ethanol amide; (vii) polycationic polymers such aspolyethylene amine; (viii) peptides that enhance transport through theBBB such as OX 26, transferrins, polybrene, histone, cationic dendrimer,synthetic peptides and polymyxin B nonapeptide (PMBN); (ix)monosaccharides such as glucose, mannose, ascorbic acid and derivativesthereof; (x) modified proteins or antibodies that undergoabsorptive-mediated or receptor-mediated transcytosis through theblood-brain barrier, such as bradykinin B2 agonist RMP-7 or monoclonalantibody to the transferrin receptor; (xi) mucoadhesive polymers such asglycerides and steroidal detergents; and (xii) Ca²⁺ chelators.
 21. Thenano-sized particle according to claim 1, wherein said at least onebiologically active agent is selected from: (i) a natural or syntheticpeptide or protein such as analgesics peptides from the enkephalinclass, insulin, insulin analogs, oxytocin, calcitonin, tyrotropinreleasing hormone, follicle stimulating hormone, luteinizing hormone,vasopressin and vasopressin analogs, catalase, interleukin-II,interferon, colony stimulating factor, tumor necrosis factor (TNF),melanocyte-stimulating hormone, superoxide dismutase, glial cell derivedneurotrophic factor (GDNF) or the Gly-Leu-Phe (GLF) families; (ii)nucleosides and polynucleotides selected from DNA or RNA molecules suchas small interfering RNA (siRNA) or a DNA plasmid; (iii) antiviral andantibacterial; (iv) antineoplastic and chemotherapy agents such ascyclosporin, doxorubicin, epirubicin, bleomycin, cisplatin, carboplatin,vinca alkaloids, e.g. vincristine, Podophyllotoxin, taxanes, e.g. Taxoland Docetaxel, and topoisomerase inhibitors, e.g. irinotecan, topotecan.22. The nano-sized particle according to claim 1, having a sphericalshape and a size of less than 200 nm, preferably less than 100 nmdiameter.
 23. The nano-sized particles according to claim 22 comprisingat least one bolaamphiphile selected from the herein designatedDerivative 1, Derivative 2, Derivative 3 and Derivative 4, an activeagent selected from leu-enkephalin, carboxyfluorescein, ¹²⁵I-GDNF, andovalbumin and at least one additive selected from vernolyl chitosan,Derivative 5, PEG-vernonia conjugate, cholesterol and cholesterylhemisuccinate.
 24. The bolaamphiphiles herein designated Derivative 1,Derivative 2, Derivative 3, Derivative 5, Derivative 6, Derivative 7,Derivative 8, Derivative 9 and Derivative
 10. 25. A pharmaceuticalcomposition comprising a nano-sized particle according to claim 1 and apharmaceutically acceptable carrier.
 26. (canceled)
 27. (canceled)
 28. Amethod for treatment of a disease or disorder selected from: (i) adisease or disorder associated with the CNS, particularlyneurological/neurodegenerative diseases or disorders such as Parkinson'sdisease, Alzheimer's disease or multiple sclerosis; (ii) cancer such asbreast cancer and brain tumors; (iii) diabetes; (iv) an immunodeficiencydisease; and (v) viral and bacterial infections, comprisingadministering to an individual in need thereof a nano-sized particleaccording to claim 1, optionally together with a suitable peripheralenzyme inhibitor to prevent premature disruption of the nano-sizedparticle.
 29. The nano-sized particles according to claim 1, comprisingat least one of the bolaamphiphile herein designated Derivative 1 andDerivative 4 or a mixture thereof, and siRNA.
 30. The pharmaceuticalcomposition according to claim 25, wherein said nano-sized particlecomprises at least one of the bolaamphiphile herein designatedDerivative 1 and Derivative 4 or a mixture thereof, and siRNA.